Monoclonal antibodies to progastrin and their uses

ABSTRACT

The present disclosure is directed to progastrin monoclonal antibodies, fragments thereof, compositions comprising progastrin monoclonal antibodies, and methods of making and using progastrin monoclonal antibodies and compositions thereof. The present disclosure is directed to methods of treating colorectal cancer with progastrin monoclonal antibodies and compositions comprising progastrin monoclonal antibodies or fragments thereof. The present disclosure is further directed to methods comprising detection of progastrin, including methods of diagnosing colorectal cancer and methods of monitoring efficacy of anti-cancer therapy in subjects suffering from colorectal cancer.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) ofprovisional application No. 61/252,625, filed Oct. 16, 2009, thecontents of all which are incorporated herein by reference in theirentirety.

2. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

None.

3. PARTIES TO A JOINT RESEARCH AGREEMENT

None.

4. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing submitted concurrently herewith under 37 CFR §1.821in a computer readable form (CRF) via EFS-Web as file nameSequence_(—)001US.txt is incorporated herein by reference. Theelectronic copy of the Sequence Listing was created on Oct. 12, 2010,with a file size of 76,910 bytes.

5. FIELD OF INVENTION

The present disclosure is directed to, among other things, monoclonalantibodies to progastrin, compositions and methods for making suchantibodies, and methods of using such antibodies, for example in thediagnosis and/or treatment of colorectal cancer.

6. BACKGROUND

Colorectal Cancer (CRC) is a major public health issue, affecting morethan 1,000,000 people each year and accounting for more than 500,000deaths each year. CRC is the second leading cause of death due tocancer. In the United States alone, for 2009, approximately 147,000 newcases and over 49,900 deaths due to CRC were reported. There are threeforms of CRC: sporadic CRC; hereditary non-polyposis colon cancer(HNPCC), caused by germline mutations in DNA mismatch repair genes; andfamilial adenomatous polyposis (FAP), due to germline mutations in theAPC gene. Sporadic CRC accounts for nearly 85% of cases, while HNPCCaccounts for about 5% and FAP accounts for about 1% (Heyer et al., 1999,Oncogene 18:5325-5333).

Clinical management of CRC typically involves surgical resection oftumors often accompanied by chemotherapy. Presently, about 50% of CRCpatients die within five years of diagnosis. The lack of reliablescreening tests and the ineffectiveness of currently available therapiesare major causes of the high mortality rate. There is an urgent need fornew clinical approaches for diagnosing CRC, as well as for treatmentseffective against colorectal cancer tumors that have minimal adverseeffects on otherwise healthy tissue.

7. SUMMARY

The present application provides compositions and methods useful fordiagnosing and/or treating colorectal cancer (CRC) in animals, includinghumans. The various inventions described in the application are based,in part, on the applicants' discovery of monoclonal antibodies thatspecifically bind progastrin (PG), for example, human progastrin (hPG),a polypeptide produced by CRC tumor cells, and that exhibitantiproliferative properties in in vitro models of CRC.

Progastrin is produced by colorectal tumor cells and is thought tostimulate proliferation of these cells by triggering a signaltransduction pathway that blocks the cells' normal differentiationprocesses, including those processes that lead to cell death. Depletionof the gastrin gene transcript that encodes progastrin induces celldifferentiation and programmed cell death in tumor cells in in vitro andin vivo CRC models, reducing tumor cell proliferation. While notintending to be bound by any theory of operation, through binding of PG,anti-hPG antibodies are thought to block or inhibit its ability tointeract with its signaling partner(s). This, in turn, inhibits a signaltransduction pathway in colorectal tumor cells that would otherwise leadto proliferation.

Accordingly, in one aspect, the present disclosure provides monoclonalantibodies that specifically bind PG, for example hPG, but not otherproducts of the gastrin gene. Referring to FIG. 1, the gastrin gene istranslated into a 101-amino acid polypeptide, called pre-progastrin,which contains a signal sequence (underlined) that is cleaved, givingrise to progastrin, an 80-amino-acid polypeptide. Progastrin, in turn,is cleaved to generate a 34-amino-acid product, corresponding toresidues 38 to 71 of progastrin, which is then extended at its carboxyterminus with a glycine residue, generating glycine-extended G34(“G34-Gly”). A by-product of this cleavage is a 5-amino-acid peptide,called the C-terminal flanking peptide, or CTFP, which includes residues75 to 80 of progastrin. G34-Gly is then further cleaved to generate a 17residue polypeptide corresponding in sequence to residues 55 to 71 ofprogastrin and referred to as G17-Gly. Removal of the C-terminalglycines of G34-Gly and G17-Gly, followed by C-terminal amidation,yields G34 and G17, respectively, both of which are C-terminal amidated.Thus, while the first 37 residues of progastrin are unique to it (i.e.not present in its processing products, such as G34, G34-Gly, G17,G17-Gly, or CFTP), residues 38 to 80 of PG are also present inpost-translational products of the gastrin gene.

Applicants have discovered that, while anti-PG monoclonal antibodies canbe raised using methods known to those of skill, the selection ofantigen is important. Not all antigens derived from hPG stimulateproduction of monoclonal antibodies that specifically bind hPG underphysiological conditions. As described below, various antigens used toraise polyclonal antibodies to hPG, such as full length recombinanthuman progastrin (see, e.g., Singh WO 08/076,454) and peptidescorresponding to the last ten amino acids at the C-terminal end of hPG(see, e.g., Hollande WO 07/135,542), failed to generate anti-hPGmonoclonal antibodies. Applicants, however, have discovered antigenic N-and C-terminal sequences within the hPG sequence that can be used togenerate monoclonal antibodies that specifically bind hPG. Quitesurprisingly, applicants have discovered that it is not necessary tolimit the antigen sequences to stretches of the PG sequence that areunique to it to obtain monoclonal antibodies that specifically bind PGand not the other gastrin gene-derived products. Peptide antigens havingsequences in common with other products of the gastrin gene, for exampleG17, G34, and CTFP, yielded monoclonal antibodies that not only bindhPG, but bind it specifically.

Applicants have generated monoclonal antibodies using antigens derivedfrom different regions of the hPG molecule. Monoclonal anti-PGantibodies obtainable using a peptide antigen having a sequencecorresponding to an N-terminal region of hPG, and/or that bind to anN-terminal region of hPG, are referred to herein as “N-terminal anti-hPGmonoclonal antibodies.” A specific exemplary antigenic region that canbe used to construct an immunogen useful for obtaining N-terminalanti-PG monoclonal antibodies corresponds to residues 1 to 14 of hPG:SWKPRSQQPDAPLG (SEQ ID NO:25). Exemplary immunogens including thisantigen useful for obtaining N-terminal anti-PG monoclonal antibodiesare described in Table 1A and the Examples section.

Monoclonal anti-PG antibodies obtainable using a peptide antigen havinga sequence corresponding to a C-terminal region of hPG, and/or that binda C-terminal region of hPG, are referred to herein as “C-terminalanti-hPG monoclonal antibodies.” A specific exemplary antigenic regionthat can be used to construct an immunogen useful for obtainingC-terminal anti-PG monoclonal antibodies corresponds to residues 55 to80 of hPG: QGPWLEEEEEAYGWMDFG RRSAEDEN (SEQ ID NO:27). Exemplaryimmunogens including this antigen useful for obtaining C-terminalanti-PG monoclonal antibodies are described in Table 1B and the Examplessection.

For some uses, it is desirable to have anti-hPG monoclonal antibodieswith high affinity to hPG. For certain uses, such as therapeutic uses,an affinity of at least about 100 nM is desirable, although antibodieshaving greater affinities, for example affinities of at least about 90nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM, 0.01 nM, or evengreater, may be desirable. The various specific exemplary anti-PGmonoclonal antibodies disclosed herein exhibit affinities ranging from10⁻⁶ to 10⁻¹²M (see Table 6). An anti-PG monoclonal antibody having anaffinity especially suited for a particular desired application can bereadily selected from amongst these, or generated or designed using thevarious immunogens, complementarity determining region (CDR) sequences,variable heavy (V_(H)) and variable light (V_(L)) chain sequences andmethods described herein. The affinity of any particular anti-PGmonoclonal antibody can be determined using techniques well known in theart or described herein, such as for example, ELISA, isothermaltitration calorimetry (ITC), BIAcore, or fluorescent polarization assay.

hPG is a relatively small polypeptide, being only 80 amino acids inlength. It would have been expected that any monoclonal antibody thatspecifically binds hPG with a relatively high affinity (e.g., at leastabout 10 nM) would interfere with PG's ability to interact with itssignaling partner(s), and, as a result, inhibit proliferation of CRCcells. However, Applicants have discovered that not all anti-PGmonoclonal antibodies are neutralizing (i.e., not all monoclonalantibodies that bind PG interfere with or inhibit its biologicalsignaling activity). Indeed, Applicants have discovered that someanti-PG monoclonal antibodies, despite exhibiting high specificity andhigh affinity for PG, do not neutralize PG. For example, anti-hPG MAb14binds hPG with a K_(D) of about 6 pM but does not inhibit the growth ofCRC cells in vitro as detailed in the Examples section below. Whilenon-neutralizing monoclonal antibodies that specifically bind hPG areuseful for diagnostic purposes, those anti-hPG monoclonal antibodiesthat neutralize PG are particularly suited for therapeutic applicationsto treat CRC.

As used herein, a “neutralizing anti-hPG monoclonal antibody” is ananti-hPG monoclonal antibody that yields a statistically significantreduction in the number of live CRC cells in a test sample treated withthe anti-hPG monoclonal antibody as compared to a control sample treatedwith a non-specific monoclonal antibody. A specific assay for assessingthe ability of any particular anti-hPG monoclonal antibody to neutralizehPG is described in the Detailed Description section below. Thoseanti-hPG monoclonal antibodies that exhibit at least about a 50%reduction in the number of live cells in this assay are believed to beespecially useful in treating CRC, although anti-hPG monoclonalantibodies exhibiting lower levels of neutralizing activity, forexample, a statistically significant reduction of 40%, 30%, 20%, 15%, oreven 10% in the number of live cells in this assay are expected toprovide therapeutic benefits.

Accordingly, in some embodiments, the anti-PG monoclonal antibodies areneutralizing anti-PG monoclonal antibodies. It has been discovered thatthe ability of an anti-PG monoclonal antibody to neutralize PG is notepitope dependent. As exemplified in the Examples section, bothN-terminal and C-terminal anti-PG antibodies have neutralizing activity.Thus, in some embodiments the neutralizing anti-PG monoclonal antibodiesare N-terminal neutralizing antibodies, in other embodiments, theanti-PG monoclonal antibodies are C-terminal neutralizing antibodies.

Epitope mapping reveals that N-terminal anti-PG monoclonal antibodies donot all bind the same epitope, even when raised against the sameimmunogen. The same is true of C-terminal anti-hPG monoclonalantibodies. The epitopes bound by exemplary N-terminal and C-terminalanti-hPG monoclonal antibodies, as identified via alanine scanning andSPOT technique, are provided in Examples section, in Tables 8 and 9.

In some embodiments, the anti-hPG monoclonal antibodies bind an epitopeincluding an amino acid sequence corresponding to an N-terminal portionof hPG. In specific embodiments, N-terminal anti-hPG monoclonalantibodies bind an epitope that includes residues 10 to 14 of hPG (SEQID NO:28), residues 9 to 14 of hPG (SEQ ID NO:29), residues 4 to 10 ofhPG (SEQ ID NO:30), residues 2 to 10 of hPG (SEQ ID NO:31), or residues2 to 14 of hPG (SEQ ID NO:32).

In some embodiments, the anti-hPG monoclonal antibodies bind an epitopeincluding an amino acid sequence corresponding to a portion of aC-terminal region of hPG. In specific embodiments, C-terminal anti-hPGmonoclonal antibodies bind an epitope that includes residues 71 to 74 ofhPG (SEQ ID NO:33), residues 69 to 73 of hPG (SEQ ID NO:34), residues 76to 80 of hPG (SEQ ID NO:35), or residues 67 to 74 of hPG (SEQ ID NO:36).

It is expected that corresponding CDRs and/or V_(H) and V_(L) chains ofanti-hPG monoclonal antibodies that bind approximately the same epitopescould be interchanged to yield new anti-hPG monoclonal antibodies. Forexample, as noted in Table 9, exemplary anti-hPG monoclonal antibodiesMAb 5 and MAb 6 bind the same epitope. An anti-hPG monoclonal antibodycan be designed that includes, in its V_(L) chain, various combinationsof the V_(L) CDRs of these two antibodies, and/or in its V_(H) chainvarious combinations of the V_(H) CDRs of these two antibodies. As aspecific example, to illustrate the various combinations possible, suchan antibody could include in its V_(L) chain, CDRs 1 and 2 of MAb 5(V_(L) CDR1.5 and V_(L) CDR2.5, respectively) and CDR 3 of MAb 6 (V_(L)CDR3.6), and in its V_(H) chain, CDR 1 of MAb 6 (V_(H) CDR1.6) and CDRs2 and 3 of MAb 5 (V_(H) CDR2.5 and V_(H) CDR3.5, respectively).

Several anti-hPG monoclonal antibodies having high specificity andaffinity for hPG and that exhibit good anti-tumor activity in in vitroassays have been identified, and in some instances the sequences oftheir CDRs, sequences of their V_(H) and V_(L) chains, and/or sequencesof proposed V_(H) and V_(L) chains for humanized versions, determined.Several hybridomas have also been deposited. All of these exemplaryanti-hPG monoclonal antibodies, as well as other specific embodiments ofanti-hPG monoclonal antibodies useful in the various kits and methodsdescribed herein, for example monoclonal antibodies that compete forbinding PG with a reference antibody, are described in more detail inthe Detailed Description section.

Anti-hPG monoclonal antibodies of the disclosure include antibodies thatcompete with a reference anti-hPG monoclonal antibody for binding hPG.The reference anti-hPG monoclonal antibody may be any of the anti-hPGmonoclonal antibodies described herein. Non-limiting examples include:antibodies comprising three V_(L) CDRs and three V_(H) CDRs as describedherein; antibodies comprising a V_(H) chain and a V_(L) chain havingamino acid sequences as set forth herein; antibodies comprisinghumanized heavy and light chain polypeptides as set forth herein;antibodies produced by any one of the hybridomas disclosed herein;antibodies that bind to an epitope within hPG as disclosed herein.

The anti-PG monoclonal antibodies described herein can be in the form offull-length antibodies, multiple chain or single chain antibodies,fragments of such antibodies that selectively bind PG (including but notlimited to Fab, Fab′, (Fab′)₂, Fv, and scFv), surrobodies (includingsurrogate light chain construct), single domain antibodies, humanizedantibodies, camelized antibodies and the like. They also can be of, orderived from, any isotype, including, for example, IgA (e.g., IgA1 orIgA2), IgD, IgE, IgG (e.g. IgG1, IgG2, IgG3 or IgG4), or IgM. In someembodiments, the anti-PG antibody is an IgG (e.g. IgG1, IgG2, IgG3 orIgG4).

Anti-PG monoclonal antibodies can be of human or non-human origin.Examples of anti-PG antibodies of non-human origin include but are notlimited to, those of mammalian origin (e.g., simians, rodents, goats,and rabbits). Anti-PG monoclonal antibodies for therapeutic use inhumans are preferably humanized.

In another aspect, the present disclosure provides nucleic acids capableof being used to produce anti-PG monoclonal antibodies. Nucleic acidsencoding immunoglobulin light chain and heavy chain polypeptides for theanti-hPG monoclonal antibodies described herein, and vectors comprisingthe nucleic acids are provided. Additionally, prokaryotic and eukaryotichost cells transformed with such vectors are provided herein, as well aseukaryotic, e.g., mammalian, host cells engineered to express the lightand heavy chain polypeptides of the anti-hPG monoclonal antibodies areprovided. Also provided are host cells capable of expressing both lightand heavy chains and secreting the monoclonal antibodies describedherein into the medium in which the host cells are cultured. In someembodiments, the host cell is a hybridoma. Methods of producing anti-hPGmonoclonal antibodies by culturing host cells are also provided.

Neutralizing anti-PG monoclonal antibodies, such as anti-hPG monoclonalantibodies, bind PG and block PG-dependent signaling, resulting in theinhibition of PG-induced responses in CRC tumor cells. Accordingly, alsoprovided are methods of inhibiting PG-induced responses of CRC cells,which includes repression of cell differentiation, repression of celldeath, and/or stimulation of cell proliferation. Generally, the methodcomprises contacting a CRC cell with, or exposing a cell population to,a neutralizing anti-PG monoclonal antibody in an amount effective toinhibit one or more PG-induced responses of CRC cells. The method can becarried out in vitro or in vivo, by administering a neutralizinganti-hPG monoclonal antibody to the environment containing CRC cells,which could be cell culture or in a tumor.

The neutralizing anti-PG monoclonal antibodies described herein inhibitPG-dependent proliferation of CRC tumor cells, making them usefultherapeutic agents for the treatment of colorectal cancer. Accordingly,also provided are pharmaceutical compositions comprising a neutralizinganti-PG monoclonal antibody and methods of using the neutralizinganti-PG monoclonal antibodies and/or pharmaceutical compositions thereofto treat CRC. The pharmaceutical compositions can be formulated for anyconvenient route of administration, including, e.g., parenteral,subcutaneous or intravenous injection, and will typically include aneutralizing anti-hPG monoclonal antibody, and one or more acceptablecarriers, excipients, and/or diluent suitable for the desired mode ofadministration, and can include other optional components as will bedescribed further in the Detailed Description section. For therapeuticuses, the compositions can be packaged in unit dosage form for ease ofuse.

The treatment methods generally comprise administering to a subject inneed of treatment, for example a subject diagnosed with CRC, an amountof a neutralizing anti-PG monoclonal antibody and/or pharmaceuticalcomposition thereof effective to provide a therapeutic benefit.Therapeutic benefit, described below in more detail, includes anyamelioration of CRC, for example, slowing or halting the progression ofCRC, reducing the severity of CRC, inhibiting the growth of CRC tumorsor the proliferation of CRC cells, reducing the size of CRC tumors,and/or reducing PG serum levels in CRC patients. The subject can be ahuman or non-human, including a domesticated animal (e.g., cat, dog,cow, pig, horse) or a non-domesticated animal. Preferably, the anti-PGmonoclonal antibody is specific to the PG of the species being treated.For example, an anti-hPG antibody is administered to a human patient, ananti-dog PG antibody is administered to a canine patient, and the like.Subjects in whom anti-hPG monoclonal antibody therapy is useful can be:patients in any stage of disease progression (e.g., CRC Stage 0, I, II,III, or IV), patients who have received CRC therapy (e.g., chemotherapy,radiation therapy, surgical resection) or patients who are receivingother therapy for CRC.

Treatment with anti-PG monoclonal antibodies as described herein can becombined with, or adjunctive to, other therapy. Non-limiting examples ofother therapy for CRC include chemotherapeutic treatment, radiationtherapy, surgical resection, and antibody therapy, as described herein.In a specific example, anti-hPG monoclonal antibodies are administeredin combination with chemotherapeutic agents. In another specificexample, anti-hPG monoclonal antibodies are administered adjunctive tosurgical resection. The anti-PG monoclonal antibodies can also be usedin combination with one another.

Individuals with CRC tumors frequently have elevated levels ofcirculating PG (e.g., in serum or plasma). Accordingly, anti-hPGmonoclonal antibodies can be used to detect PG levels for purposes ofdiagnosing CRC. Additionally, in patients already diagnosed with CRC,anti-hPG monoclonal antibodies can be used to select subjects suitablefor receiving anti-PG therapy, or monitoring treatment efficacy. Asdisclosed herein, a method of diagnosing colorectal cancer in a subjectcomprises determining whether the amount of progastrin in a sample fromthe subject, for example a blood sample or a serum sample, measuredusing an anti-hPG monoclonal antibody according to the presentdisclosure, is above a threshold level. In a specific embodiment, thethreshold level is 50 pM. In some embodiments, two anti-PG antibodiesare used, one that recognizes a C-terminal region of PG and another thatrecognizes an N-terminal region of PG. In this embodiment, one or bothof the N-terminal or C-terminal antibodies is an anti-PG monoclonalantibody as described herein. Preferably, N-terminal and C-terminalanti-PG monoclonal antibodies are used. The antibodies may be, but neednot be, neutralizing.

For purposes of monitoring treatment efficacy, anti-PG monoclonalantibodies can be used to determine whether the level of progastrin isdecreasing over time in samples from a subject who has been or is beingtreated for CRC by comparing the amount of PG in samples taken atdifferent times. The specific embodiments of anti-PG antibodiesdescribed in the preceding paragraph can also be used in this assay.

Also provided are kits suitable for carrying out the various diagnostic,monitoring, and other methods described herein. Such kits will typicallycomprise an anti-PG monoclonal antibody as described herein and,optionally, additional anti-PG antibodies and/or reagents suitable forperforming the specific assay. In some embodiments, one or more anti-PGantibodies included in the kit is labeled with a detectable label, suchas a fluorophore. In a specific embodiment, the kit includes an anti-PGantibody that specifically binds an N-terminal region of PG, an anti-PGantibody that specifically binds a C-terminal region of PG andoptionally, reagents suitable for performing a diagnostic assay, wherethe N-terminal specific antibody is an N-terminal anti-PG monoclonalantibody as described herein and/or the C-terminal specific antibody isa C-terminal anti-PG monoclonal antibody as described herein.

The features and advantages of the various inventions described hereinwill become further apparent from the following detailed description ofexemplary embodiments thereof.

8. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides amino acid sequences of pre-progastrin (wherein thesignal peptide is underlined), progastrin and products of progastrinprocessing including G34, G34-Gly, G17, G17-Gly, and the C-terminalflanking peptide, CTFP.

FIG. 2 provides polypeptide, and corresponding polynucleotide, sequencesof V_(H) and V_(L) chains for exemplary murine anti-hPG monoclonalantibodies: anti-hPG MAb 3 (SEQ ID NOs:16, 12, 17 and 13, respectively,in order of appearance) (FIG. 2A, 2B), anti-hPG MAb 4 (SEQ ID NOs:18,14, 19 and 15, respectively, in order of appearance) (FIG. 2C, 2D),anti-hPG MAb 8 (SEQ ID NOs:67, 59, 71 and 63, respectively, in order ofappearance) (FIG. 2E, 2F), anti-hPG Mab 13 (SEQ ID NOSs:68, 60, 72 and64, respectively, in order of appearance) (FIG. 2G, 2H), anti-hPG MAb 16(SEQ ID NOs:69, 61, 73 and 65, respectively, in order of appearance)(FIG. 2I, 2J), and anti-hPG MAb 19 (SEQ ID NOs:70, 62, 74 and 66,respectively, in order of appearance) (FIG. 2K, 2L), in which the threeCDRs of each chain are underlined.

FIG. 3A-C provide graphs illustrating relative binding affinities(measured as absorbance at 492 nm) at increasing antibody concentrations(μg/mL) of exemplary murine anti-hPG monoclonal antibodies, MAbs 1-4(FIG. 3A); MAbs 5-14 and 20-23 (FIG. 3B); and MAbs 3 and 15-19 (FIG.3C).

FIG. 4 provides a graph illustrating the ratio of absorbance (opticaldensity) at 280 nm and 330 nm for four different exemplary murineanti-hPG monoclonal antibodies as compared to a control sample of bovineserum albumin (arbitrary units).

FIG. 5A-C provide graphs illustrating the binding of 23 differentexemplary murine anti-hPG monoclonal antibodies to 25 or 50 ng hPG ascompared to: buffer alone (negative control), 250 ng KLH (negativecontrol), and peptides derived from the gastrin gene (50 and 250 ng ofCTFP, G17, or G17-Gly (referred to in the figure as “G-Gly”), asindicated. FIG. 5A shows the binding of anti-hPG MAbs 1-4, FIG. 5B showsthe binding of anti-hPG MAbs 5-14 and 21-23, and FIG. 5C shows thebinding of anti-hPG MAbs 3 and 15-20.

FIG. 6 provides a graph illustrating the binding of a polyclonalanti-hPG antibody that binds an N-terminal region of hPG at increasingconcentrations of anti-hPG MAb3.

FIG. 7 provides graphs illustrating proliferation of representative CRCcell lines treated with anti-hPG monoclonal antibodies as follows:SW480, HCT-116, LS174T, as indicated, treated with exemplary anti-hPGmonoclonal antibodies MAb 3 and MAb 4 (FIG. 7A, 7B, 7C, respectively,showing the change in number of live cells at the end of treatmentrelative to the beginning of treatment (T0) with the indicatedantibody), or an anti-hPG polyclonal antibody (FIG. 7D, 7E, 7F,respectively, showing the change in number of live cells at the end oftreatment relative to the beginning of treatment (T0) with antibody);proliferation of CRC cell line SW620 treated with anti-hPG MAb 5 to MAb23 (FIG. 7G, showing live anti-hPG-treated cells as a percentage of thenumber of control antibody-treated cells at the end of treatmentrelative to the beginning of treatment (T0)); proliferation of LS174Tcells treated with anti-hPG MAb 8, 13, 14, 16, and 19 (FIG. 7H, showinglive anti-hPG-treated cells as a percentage of the number of controlantibody-treated cells at the end of treatment relative to the beginningof treatment (T0)); and proliferation of HCT-116 cells treated withanti-hPG monoclonal antibodies MAb 8, 13, 14, 16, 19 (FIG. 7I, showinglive anti-hPG-treated cells as a percentage of the number of controlantibody-treated cells at the end of treatment relative to the beginningof treatment (T0)).

FIG. 8 provides a graph illustrating the number of live LS174T cells at48 hours after 4 treatments with a control monoclonal antibody, anti-hPGMAb 8 (5 μg/mL), anti-hPG MAb 8 pre-incubated with hPG, the controlantibody pre-incubated with hPG, or hPG alone.

FIG. 9 provides graphs illustrating the number of tumors per mouse (FIG.9A) and average tumor length and height (FIG. 9B) in mice treated withanti-hPG antibodies as compared to a control polyclonal antibody.

9. DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS 9.1. DetailedDescription

Progastrin (PG) was first identified as the precursor to gastrin, a gutpeptide hormone that stimulates gastric acid secretion. Gastrin existsin a number of different molecular forms (G17, G34, glycine-extendedG17, glycine-extended G34) derived from progastrin. See FIG. 1. Thegastrin gene encodes a 101-amino acid product, preprogastrin. A firstcleavage removes a 21-amino-acid residue signal peptide (underlined inFIG. 1) and results in PG, an 80 amino acid peptide. The understood,known polypeptide sequence of human PG (hPG) is provided in SEQ IDNO:20. As illustrated in FIG. 1, the amino acid residues of hPG arenumbered from 1 to 80, with the amino-most residue being position 1.Sequences within the first 40 amino acids of progastrin are referred toas “N-terminal,” while sequences falling within residue 41 to 80 arereferred to as “C-terminal.”

Recent studies have shown that progastrin levels are elevated inpatients with CRC. Under normal physiological conditions, progastrinaccounts for less than 10% of total secreted peptide in humans. Incolorectal cancer, progastrin levels are significantly elevated in bothplasma and tumor tissue, possibly as a result of increased expression ofthe gastrin gene coupled with incomplete processing of the gene product.One study showed significantly higher serum progastrin levels in CRCpatients as compared to control patients but no such difference for themore processed forms of gastrin (Siddheshwar et al., 2001, Gut48:47-52). In CRC tumor samples tested, 80-100% of samples showedincreased PG levels. See, e.g., Ciccotosto et al., 1995,Gastroenterology 109:1142-1153; Baldwin et al., 1998, Gut 42:581-584;Van Solinge, 1993, Gastroenterology 104:1099-1107. The role of PG in CRChas been further substantiated by experiments showing that miceexpressing recombinant human PG treated with the carcinogen azoxymethanehad significantly greater numbers of aberrant crypt foci, adenomas, andadenocarcinomas in the colon as compared to wild type mice or miceexpressing amidated gastrins (Singh et al. 2000, Gastroenterology119:162-171).

Recently, Hollande et al., demonstrated that progastrin stimulates thebeta-catenin/Tcf4 pathway by repressing ICAT, a negative regulator ofbeta-catenin/Tcf4 signaling, and that blocking progastrin leads to denovo expression of ICAT. See WO 2007/135542. While not intending to bebound by any theory of operation, it is believed that blockingprogastrin signaling leads to repression of beta-catenin/Tcf4-inducedproliferation as a result of increased ICAT expression. In the absenceof continued PG-dependent signaling, cell proliferation is inhibited,and cell differentiation and/or cell death (including apoptosis) istriggered.

Despite the urgent need for new clinical approaches to the treatment anddiagnosis of CRC, the evidence that PG stimulates proliferation of CRCtumor cells, and despite the increased focus on monoclonal antibodytherapies in the treatment of cancer, to date, there are no reportsdemonstrating any monoclonal antibody capable of blocking PG-dependenttumor cell proliferation, or even binding PG. Such antibodies, presentedherein for the first time, proved difficult to develop. As a firstchallenge, applicants found that recombinant human progastrin, which canbe used to generate polyclonal anti-hPG antibodies, failed to induce amonoclonal immunogenic response in test mice. Therefore, it wasnecessary to design immunogens using only peptide fragments of PG togenerate antibodies specific to progastrin and not other gastrin geneproducts. Even once hybridoma clones yielded antibodies that bound theantigenic peptide, it was found that binding to the peptide was notpredictive of the ability to bind PG, specifically or at all. As shownin more detail in the Examples below, many hybridomas yielded antibodiesthat bound the PG antigen peptide used in the immunogen but failed tobind PG. The present disclosure provides anti-hPG monoclonal antibodiesthat bind not only the peptide antigen against which they were raisedbut also that bind hPG specifically. Quite surprisingly, monoclonalantibodies highly specific for hPG relative to its processing products(e.g., G34, G34-Gly, G17, G17-Gly, CTFP) were obtained with antigensthat in some cases are not unique to hPG, but that included regions ofamino acid sequence common to one or more of the progatrin processingproducts. Moreover, it was also surprisingly discovered that despite therelatively small size of hPG (80 amino acids) not all anti-hPGmonoclonal antibodies, even those exhibiting a high degree of affinityand specificity for hPG, neutralize its biological activity.

Anti-hPG Monoclonal Antibodies

Applicants have discovered peptide antigens useful for raising anti-hPGmonoclonal antibodies. Peptides useful for raising anti-hPG antibodiesof the present disclosure comprise progastrin-specific sequences notfound in the more processed forms of the polypeptide, such asglycine-extended or amidated gastrins or CTFP, but can also comprisesequences that are found in processed forms of hPG. In some embodiments,anti-hPG monoclonal antibodies are raised against a peptide antigenhaving an amino acid sequence corresponding to an N-terminal region ofhPG and are designated N-terminal anti-hPG monoclonal antibodies. Aspecific exemplary antigenic region that can be used to construct animmunogen useful for obtaining N-terminal anti-PG monoclonal antibodiescorresponds to residues 1 to 14 of hPG (SWKPRSQQPDAPLG (SEQ ID NO: 25))coupled to a linker sequence. In other embodiments, the anti-hPGmonoclonal antibodies are raised against a peptide antigen having anamino acid sequence corresponding to a C-terminal region of hPG and aredesignated C-terminal anti-hPG monoclonal antibodies. A specificexemplary antigenic region that can be used to construct an immunogenuseful for obtaining C-terminal anti-PG monoclonal antibodiescorresponds to residues 55 to 80 of hPG (SEQ ID NO:27) coupled to alinker sequence. See Table 1.

Anti-PG monoclonal antibodies of the present disclosure bind PG and areuseful for detecting and isolating PG from complex mixtures.Additionally, the anti-PG monoclonal antibodies of the disclosure areuniquely suited to therapeutic and/or diagnostic applications forcolorectal cancer. In various embodiments, anti-hPG monoclonalantibodies (1) specifically bind PG versus other gastrin gene products,(2) have high affinity to hPG, (3) inhibit colorectal cancer cellproliferation in vitro and in vivo, (4) reduce tumor size and number invivo, (5) detect PG in complex mixtures containing other gastrin-genederived peptides.

The gastrin gene is expressed and extensively processed, to yieldseveral protein products that have roles in normal homeostasis.Progastrin, on the other hand, is typically not detectable in thecirculation of healthy subjects. The monoclonal antibodies of thepresent disclosure are intended to target progastrin but not otherpeptides derived from the gastrin gene. Accordingly, anti-hPG monoclonalantibodies specifically bind to progastrin, from humans and otheranimals, but not to other gastrin gene products, such as, but notlimited to, glycine-extended or amidated gastrins, or C-terminalFlanking Peptide (CTFP).

Specificity of anti-hPG monoclonal antibodies can be determined using anELISA as follows. 96-well plates are incubated overnight at 4° C. withappropriate concentration(s) of test polypeptide (e.g., 25 and 50 ngrecombinant human PG, and 50 and 250 ng CTFP or other gastrin-derivedgene products) in Phosphate-Buffered Saline (PBS), after which the wellsare washed three times with wash solution (PBS and 0.1% Tween-20), andthen incubated for 2 hours at 22° C. with 100 μL blocking solution (PBS,0.1% Tween-20, 0.1% Bovine Serum Albumin or casein hydrolysate) perwell. After blocking, the wells are washed three times and the antibodyto be assayed (test antibody) is added. 100 μl of the test antibody (at0.3 to 1 ng/mL) in PBS and 0.1% Tween-20 are added to each well. Platesare then incubated for 2 hours at 22° C., after which the test antibodysolution is discarded and replaced, after a wash step (3×100 μL washsolution, as noted above), with blocking solution containing a secondaryantibody, a goat anti-mouse IgG (Fc) antibody coupled to horseradishperoxidase. After a 1-hour incubation with secondary antibody, 100 μL ofsubstrate solution (e.g. Fast OPD, or O-Phenylenediaminedihydrochloride, available from Sigma-Aldrich Co., prepared according tomanufacturer's directions) is added to each well and incubated in thedark for 20 minutes at 22° C. The reaction is stopped by adding 50 μL of4N sulfuric acid and the amount of substrate catalyzed determined bymeasuring the optical density (O.D.) at 492 nm. Substrate conversion isproportional to the amount of primary (test) antibody bound to theantigen. Experiments are run in duplicate and OD measurements plotted asa function of antigen concentration. Test antibodies are scored asspecific for PG if the measured O.D. is between 0.2 and 1.5 for hPG andthere is no statistically significant signal above background with CTFPor any of the other gastrin-gene derived peptides, where the backgroundis the average signal from control wells containing only PBS.

Several anti-hPG monoclonal antibodies of the present disclosure werefound to be highly specific. In some embodiments, anti-hPG monoclonalantibodies exhibit 100-fold greater specificity for progastrin ascompared to the other gastrin gene products. In such embodiments,100-fold more antigen (e.g., glycine-extended or amidated gastrin) isrequired to yield the same binding as is observed when the antigen isprogastrin.

Other methods for determining binding include, but are not limited to,an immunofluorescent method, an enzyme-linked immunosorbent assay(ELISA), a radioactive material labeled immunoassay (RIA), a sandwichELISA (Monoclonal Antibody Experiment Manual (published by KodanshaScientific, 1987), Second Series Biochemical Experiment Course, Vol. 5,Immunobiochemistry Research Method, published by Tokyo Kagaku Dojin(1986)).

Anti-hPG monoclonal antibodies with high affinity for PG are desirablefor both therapeutic and diagnostic uses. For certain uses, such astherapeutic uses, an affinity of at least about 100 nM is desirable,although antibodies having greater affinities, for example affinities ofat least about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM,20 nM, 15 nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM,0.01 nM, 10 pM, 1 pM, or even greater, may be desirable. In someembodiments, the monoclonal antibodies specifically bind hPG with anaffinity in the range of about 1 pM to about 100 nM, or an affinityranging between any of the foregoing values.

Affinity of anti-hPG monoclonal antibodies for hPG can be determinedusing techniques well known in the art or described herein, such as forexample, but not by way of limitation, ELISA, isothermal titrationcalorimetry (ITC), BIAcore, Proteon, or fluorescent polarization assay.

Using antigens from N- or C-terminal regions of hPG, antibodies thatrecognize different epitopes of hPG can be generated. The epitoperecognized by a monoclonal antibody will depend on the particularantigen used to raise the antibody and can be mapped using techniquesknown to the skilled artisan, such as alanine scanning and SPOT analysis(see Examples section below). For example, epitope mapping reveals thatanti-hPG MAb 2 and MAb 4 bind the same epitope; anti-hPG MAb 1 and MAb 3bind approximately the same epitope; MAb 17, MAb 18, MAb 19, and MAb 20bind approximately the same epitope; MAb 15 and MAb 16 bindapproximately the same epitope; anti-hPG MAb 5, MAb 6, MAb 7, MAb 9, andMAb 12 bind the same epitope and bind approximately the same epitope asanti-hPG MAb 10; and anti-hPG MAb 11 and MAb 14 bind approximately thesame epitope.

Whether or not an anti-hPG monoclonal antibody recognizes a particularepitope can be determined using a competition assay as described herein,in which the epitope bound by the reference antibody is known. In someembodiments, the anti-hPG monoclonal antibody competes with a referenceantibody which binds an epitope having an amino acid sequencecorresponding to an N-terminal region of hPG. In specific embodiments,anti-hPG monoclonal antibodies compete with a reference antibody thatbinds an epitope that includes residues 10 to 14 of hPG (SEQ ID NO:28),residues 9 to 14 of hPG (SEQ ID NO:29), residues 4 to 10 of hPG (SEQ IDNO:30), residues 2 to 10 of hPG (SEQ ID NO:31), or residues 2 to 14 ofhPG (SEQ ID NO:32). In some embodiments, the anti-hPG monoclonalantibody competes with a reference antibody which binds an epitopehaving an amino acid sequence corresponding to a C-terminal region ofhPG. In specific embodiments, anti-hPG monoclonal antibodies competewith a reference antibody that binds an epitope that includes residues71 to 74 of hPG (SEQ ID NO:33), residues 69 to 73 of hPG (SEQ ID NO:34),residues 76 to 80 of hPG (SEQ ID NO:35), or residues 67 to 74 of hPG(SEQ ID NO:36).

The anti-PG monoclonal antibodies can be neutralizing. While notintending to be bound by any theory of operation, through binding of PG,neutralizing anti-hPG monoclonal antibodies are thought to block orinhibit its ability to interact with its signaling partner(s). This, inturn, inhibits a signal transduction pathway in colorectal tumor cellsthat would otherwise lead to proliferation, reduced cell differentiationand cell death. In some embodiments, neutralizing anti-PG monoclonalantibodies bind an N-terminal region of hPG. In specific embodiments,neutralizing anti-PG monoclonal antibodies compete for binding PG withanti-hPG MAb1, MAb2, MAb3, MAb4, MAb15, MAb16, MAb17, MAb18, MAb19, orMAb20. In other embodiments, neutralizing anti-PG monoclonal antibodiesbind a C-terminal region of hPG. In specific embodiments, neutralizinganti-PG monoclonal antibodies compete for binding PG with anti-hPG MAb5,MAb6, MAb7, MAb8, MAb9, MAb10, MAb11, MAb12, MAb13, MAb21, MAb22, orMAb23.

A specific test for whether an anti-PG monoclonal antibody isneutralizing can be performed as follows. CRC LS174T cells are seeded ina 6-well plate, as described in Example 7 below, at approximately 50,000cells per well. Cells are then treated at 12 hour intervals for 48 hourswith the test anti-PG monoclonal antibody or a control monoclonalantibody as noted in Example 7, at antibody concentrations of about 5μg/mL. A test antibody is defined as neutralizing in the assay, if thenumber of CRC cancer cells treated with the test antibody shows astatistically significant reduction of at least 10% in the number ofsurviving cells compared to the number of cells treated with a control,non-specific antibody, using a two-tailed Mann-Whitney test (withdifferences considered as significant when p<0.05). Total cell numbersare corrected for the number of cells at the start of the treatmentperiod, referred to as T0.

As used herein, an antibody (Ab) refers to an immunoglobulin moleculethat specifically binds to, or is immunologically reactive with, aparticular antigen, and includes polyclonal, monoclonal, geneticallyengineered and otherwise modified forms of antibodies, including but notlimited to chimeric antibodies, humanized antibodies, and antigenbinding fragments of antibodies, including e.g., Fab′, F(ab′)₂, Fab, Fv,rIgG, and scFv fragments. In various embodiments, anti-hPG monoclonalantibodies comprise all or a portion of a constant region of anantibody. In some embodiments, the constant region is an isotypeselected from: IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1,IgG2, IgG3 or IgG4), and IgM.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. A monoclonal antibodyis derived from a single clone, including any eukaryotic, prokaryotic,or phage clone, by any means available or known in the art. Monoclonalantibodies useful with the present disclosure can be prepared using awide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof. In many uses of the present disclosure, including in vivo useof the anti-hPG monoclonal antibodies in humans and in vitro detectionassays, chimeric, primatized, humanized, or human antibodies cansuitably be used.

The term “scFv” refers to a single chain Fv antibody in which thevariable domains of the heavy chain and the light chain from atraditional antibody have been joined to form one chain.

References to “V_(H)” refer to the variable region of an immunoglobulinheavy chain of an antibody, including the heavy chain of an Fv, scFv, orFab. References to “V_(L)” refer to the variable region of animmunoglobulin light chain, including the light chain of an Fv, scFv,dsFv or Fab. Antibodies (Abs) and immunoglobulins (Igs) areglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific target,immunoglobulins include both antibodies and other antibody-likemolecules which lack target specificity. Native antibodies andimmunoglobulins are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atits other end.

Anti-hPG monoclonal antibodies of the disclosure comprisecomplementarity determining regions (CDRs). CDRs are also known ashypervariable regions both in the light chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). As is known in the art, the amino acidposition/boundary delineating a hypervariable region of an antibody canvary, depending on the context and the various definitions known in theart. Some positions within a variable domain may be viewed as hybridhypervariable positions in that these positions can be deemed to bewithin a hypervariable region under one set of criteria while beingdeemed to be outside a hypervariable region under a different set ofcriteria. One or more of these positions can also be found in extendedhypervariable regions. The disclosure provides antibodies comprisingmodifications in these hybrid hypervariable positions. The variabledomains of native heavy and light chains each comprise four FR regions,largely by adopting a β-sheet configuration, connected by three CDRs,which form loops connecting, and in some cases forming part of, theβ-sheet structure. The CDRs in each chain are held together in closeproximity by the FR regions and, with the CDRs from the other chain,contribute to the formation of the target binding site of antibodies(See Kabat et al., Sequences of Proteins of Immunological Interest(National Institute of Health, Bethesda, Md. 1987).

Several anti-hPG monoclonal antibodies having high specificity andaffinity for hPG and good anti-tumor activity have been identified, andtheir CDRs and variable heavy and light chains have been sequenced.Murine heavy and light chain variable domains are referred to herein asmV_(H) and mV_(L) followed by the number of the corresponding monoclonalantibody, for example mV_(H).3 and mV_(L).3 for anti-hPG MAb3. Anti-hPGmonoclonal antibodies have three variable light chain CDRs and threevariable heavy chain CDRs, referred to as V_(H) CDR1, 2, or 3, and V_(L)CDR1, 2, or 3, respectively, followed by the number of the exemplaryanti-hPG monoclonal antibody. For example, V_(L) CDR1 of MAb 3 isdenoted V_(L) CDR1.3 and V_(H) CDR1 of MAb 3 is denoted V_(H) CDR1.3.Similarly, human heavy and light chain variable domains are referred toherein as hV_(H) and hV_(L) followed by the number of the correspondingmonoclonal antibody.

In some embodiments, anti-hPG monoclonal antibodies raised against anN-terminal portion of hPG have three variable light chain CDRs and threevariable heavy chain CDRs, wherein the V_(L) CDR1 is selected fromQSIVHSNGNTY (“V_(L) CDR 1.3”; SEQ ID NO:4), QSLVHSSGVTY (“V_(L) CDR1.4”; SEQ ID NO:10), QSLLDSDGKTY (“V_(L) CDR 1.16”; SEQ ID NO:50), andSQHRTYT (“V_(L) CDR 1.19”; SEQ ID NO:51); the V_(L) CDR2 is selectedfrom KVS (“V_(L) CDR 2.3” and (“V_(L) CDR 2.4”; SEQ ID NO:5), LVS(“V_(L) CDR 2.16”; SEQ ID NO:53), and VKKDGSH (“V_(L) CDR 2.19”; SEQ IDNO:54); the V_(L) CDR3 is selected from FQGSHVPFT (“V_(L) CDR 3.3”; SEQID NO:6), SQSTHVPPT (“V_(L) CDR 3.4”; SEQ ID NO:11), WQGTHSPYT (“V_(L)CDR 3.16”; SEQ ID NO:57), and GVGDAIKGQSVFV (“V_(L) CDR 3.19”; SEQ IDNO:58); the V_(H) CDR1 is selected from GYIFTSYW (“V_(H) CDR 1.3”; SEQID NO:1), GYTFSSSW (“V_(H) CDR 1.4”; SEQ ID NO:7), GYTFTSYY (“V_(H) CDR1.16”; SEQ ID NO:39), and GYSITSDYA (“V_(H) CDR 1.19”; SEQ ID NO:40);the V_(H) CDR2 is selected from FYPGNSDS (“V_(H) CDR 2.3”; SEQ ID NO:2),FLPGSGST (“V_(H) CDR 2.4”; SEQ ID NO:8), INPSNGGT (“V_(H) CDR 2.16”; SEQID NO:43), and ISFSGYT (“V_(H) CDR 2.19”; SEQ ID NO:44); and the V_(H)CDR3 is selected from TRRDSPQY (“V_(H) CDR 3.3”; SEQ ID NO:3),ATDGNYDWFAY (“V_(H) CDR 3.4” SEQ ID NO:9), TRGGYYPFDY (“V_(H) CDR 3.16”;SEQ ID NO:47), and AREVNYGDSYHFDY (“V_(H) CDR 3.19”; SEQ ID NO:48). SeeTable 1A.

In some embodiments, anti-hPG monoclonal antibodies raised against aC-terminal portion of hPG have three variable light chain CDRs and threevariable heavy chain CDRs, wherein the V_(L) CDR1 is selected fromKSLRHTKGITF (“V_(L) CDR 1.8”; SEQ ID NO:49) and QSLLDSDGKTY (“V_(L) CDR1.13”; SEQ ID NO:50); the V_(L) CDR2 is selected from QMS (“V_(L) CDR2.8”; SEQ ID NO:52) and LVS (“V_(L) CDR 2.13”; SEQ ID NO:53); the V_(L)CDR3 is selected from AQNLELPLT (“V_(L) CDR 3.8”; SEQ ID NO:55) andWQGTHFPQT (“V_(L) CDR 3.13”; SEQ ID NO:56); the V_(H) CDR1 is selectedfrom GFTFTTYA (“V_(H) CDR 1.8”; SEQ ID NO:37) and GFIFSSYG (“V_(H) CDR1.13”; SEQ ID NO:38); the V_(H) CDR2 is selected from ISSGGTYT (“V_(H)CDR 2.8”; SEQ ID NO:41) and INTFGDRT (“V_(H) CDR 2.13”; SEQ ID NO:42);and the V_(H) CDR3 is selected from ATQGNYSLDF (“V_(H) CDR 3.8”; SEQ IDNO:45) and ARGTGTY (“V_(H) CDR 3.13”; SEQ ID NO:46). See Table 1B.

TABLE 1A N-Terminal Anti-hPG Monoclonal Antibodies Hybridoma Immu-(Deposit Murine V_(H) Humanized V_(H) and V_(L) nogen #) MAbMurine CDR Sequences and V_(L) Sequences Sequences (projected) N143B9G11 MAb1 N1 WE5H2G7 MAb2 N2 6B5B11C10 MAb3 V_(H) CDR 1.3 GYIFTSYW(SEQ ID NO: 1) mV_(H) 3 (SEQ ID NO. 12) hV_(H) 3 (SEQ ID NO: 21)V_(H) CDR 2.3 FYPGNSDS (SEQ ID NO: 2) V_(H) CDR 3.3 TRRDSPQY(SEQ ID NO: 3) V_(L) CDR 1.3 QSIVHSNGNTY (SEQ ID NO: 4) mV_(L) 3(SEQ ID NO: 13) hV_(L) 3 (SEQ ID NO: 22) V_(L) CDR 2.3 KVS(SEQ ID NO: 5) V_(L) CDR 3.3 FQGSHVPFT (SEQ ID NO: 6) N2 20D2C3G2 MAb4V_(H) CDR 1.4 GYTFSSSW (SEQ ID NO: 7) mV_(H) 4 (SEQ ID NO: 14) hV_(H) 4(SEQ ID NO: 23) V_(H) CDR 2.4 FLPGSGST (SEQ ID NO: 8) V_(H) CDR 3.4ATDGNYDWFAY (SEQ ID NO: 9) V_(L) CDR 1.4 QSLVHSSGVTY (SEQ ID NO: 10)mV_(L) 4 (SEQ ID NO: 15) hV_(L) 4 (SEQ ID NO: 24) V_(L) CDR 2.4 KVS(SEQ ID NO: 5) V_(L) CDR 3.4 SQSTHVPPT (SEQ ID NO: 11) N2 1E9A4A4 MAb15(1-4376) N2 1E9D9B6 MAb16 V_(H) CDR 1.16 GYTFTSYY (SEQ ID NO: 39)mV_(H) 16  (SEQ ID NO: 61) hV_(H) 16a (SEQ ID NO: 84) V_(H) CDR 2.16INPSNGGT (SEQ ID NO: 43) hV_(H) 16b  (SEQ ID NO: 86) V_(H) CDR 3.16TRGGYYPFDY (SEQ ID NO: 47) hV_(H) 16c  (SEQ ID NO: 88) V_(L) CDR 1.16QSLLDSDGKTY (SEQ ID NO: 50) mV_(L) 16 (SEQ ID NO: 65) hV_(L) 16a (SEQ ID NO: 85) V_(L) CDR 2.16 LVS (SEQ ID NO: 53) hV_(L) 16b (SEQ ID NO: 87) V_(L) CDR 3.16 WQGTHSPYT (SEQ ID NO: 57) hV_(L) 16c (SEQ ID NO: 89) N2 1C8D10F5  MAb17 N2 1A7C3F11 MAb18 N2 1B3B4F11 MAb19V_(H) CDR 1.19 GYSITSDYA (SEQ ID NO: 40) mV_(H) 19  (SEQ ID NO: 62)hV_(H) 19a (SEQ ID NO: 90) V_(H) CDR 2.19 ISFSGYT (SEQ ID NO: 44)hV_(H) 19b  (SEQ ID NO: 92) V_(H) CDR 3.19 AREVNYGDSYHFDY(SEQ ID NO: 48) hV_(H) 19c  (SEQ ID NO: 94) V_(L) CDR 1.19 SQHRTYT(SEQ ID NO: 51) mV_(L) 19 (SEQ ID NO: 66) hV_(L) 19a  (SEQ ID NO: 91)V_(L) CDR 2.19 VKKDGSH (SEQ ID NO: 54) hV_(L) 19b  (SEQ ID NO: 93)V_(L) CDR 3.19 GVGDAIKGQSVFV (SEQ ID NO: 58) hV_(L) 19c  (SEQ ID NO: 95)N2 1C11E5E8  MAb20 Immunogen N1 = SWKPRSQQPDAPLG Ahx Cys BSA, alsorepresented as (SEQ ID NO: 25) Ahx Cys BSA Immunogen N2 = SWKPRSQQPDAPLGAhx Cys KLH, also represented as (SEQ ID NO: 25) Ahx Cys KLH In Table1A, all amino acid sequences are represented using conventional N→Corientation. For each immunogen, the progastrin peptide was synthesizedwith a linker of one aminohexanoic acid (Ahx) residues followed by acysteine, which was then conjugated to a either a bovine serum albumin(“BSA”) or keyhole limpet hemocyanin (“KLH”) carrier.

TABLE 1B C-Terminal Anti-hPG Monoclonal Antibodies Hybridoma Immu-(Deposit Murine V_(H) Humanized V_(H) and V_(L) nogen #) MAbMurine CDR Sequences and V_(L) Sequences Sequences (projected) C11B4A11D11 MAb5 (1-4371) C1 1B6A11F2 MAb6 (1-4372) C1 1B11E4B11  MAb7(1-4373) C1 1C10D3B9 MAb8 V_(H) CDR 1.8 GFTFTTYA (SEQ ID NO: 37)mV_(H).8 (SEQ ID NO: 59) hV_(H) 8a (SEQ ID NO: 75) V_(H) CDR 2.8ISSGGTYT (SEQ ID NO: 41) hV_(H) 8b (SEQ ID NO: 77) V_(H) CDR 3.8ATQGNYSLDF (SEQ ID NO: 45) hV_(H) 8c (SEQ ID NO: 79) V_(L) CDR 1.8KSLRHTKGITF (SEQ ID NO: 49) mV_(L).8 (SEQ ID NO: 63) hV_(L) 8a(SEQ ID NO: 76) V_(L) CDR 2.8 QMS (SEQ ID NO: 52) hV_(L) 8b(SEQ ID NO: 78) V_(L) CDR 3.8 AQNLELPLT (SEQ ID NO: 55) hV_(L) 8c(SEQ ID NO: 76) C1 1D8F5B3 MAb9 C1 1E1C7B4 MAb10 C1 2B4C8C8 MAb11(1-4374) C1 2B11E6G4  MAb12 (1-4375) C1 2C6C3C7 MAb13 V_(H) CDR 1.13GFIFSSYG (SEQ ID NO: 38) mV_(H).13 (SEQ ID NO: 60) hV_(H).13a(SEQ ID NO: 80) V_(H) CDR 2.13 INTFGDRT (SEQ ID NO: 42) hV_(H).13b(SEQ ID NO: 82) V_(H) CDR 3.13 ARGTGTY (SEQ ID NO: 46) V_(L) CDR 1.13QSLLDSDGKTY (SEQ ID NO: 50) mV_(L).13 (SEQ ID NO: 64) hV_(L) 13a(SEQ ID NO: 81) V_(L) CDR 2.13 LVS (SEQ ID NO: 53) hV_(L) 13b(SEQ ID NO: 83) V_(L) CDR 3.13 WQGTHFPQT (SEQ ID NO: 56) C1 2H9F4B7MAb14 C2 1F11F5E10  MAb21 C2 1F11F5G9 MAb22 C2 1A11F2C9 MAb23 ImmunogenC1 = KLH Cys Ahx Ahx QGPWLEEEEEAYGWMDFGRRSAEDEN, also represented as KLHCys Ahx Ahx (SEQ ID NO: 27) Immunogen C2 = DT Cys Ahx AhxQGPWLEEEEEAYGWMDFGRRSAEDEN, also represented as DT Cys Ahx Ahx (SEQ IDNO: 27) In Table 1B, all amino acid sequences are represented usingconventional N→C orientation. For each immunogen, the progastrin peptidewas synthesized with a linker of two aminohexanoic acid (Ahx) residuesfollowed by a cysteine, which was then conjugated to a either a keyholelimpet hemocyanin (“KLH”) or a diphtheria toxin (“DT”) carrier.

In some embodiments, the CDRs of the V_(H) chain of the anti-hPGmonoclonal antibody are V_(H)CDR1.3, V_(H)CDR2.3 and V_(H)CDR3.3. In aspecific embodiment, the V_(H) chain of the anti-hPG monoclonal antibodyhas an amino acid sequence corresponding to mV_(H).3 (SEQ ID NO:12). SeeFIG. 2A.

In some embodiments, the CDRs of the V_(L) chain of the anti-hPGmonoclonal antibody are V_(L)CDR1.3, V_(L)CDR2.3 and V_(L)CDR3.3. In aspecific embodiment, the V_(L) chain of the anti-hPG monoclonal antibodyhas an amino acid sequence corresponding to mV_(L).3 (SEQ ID NO:13). SeeFIG. 2B.

In some embodiments, the CDRs of the V_(H) chain of the anti-hPGmonoclonal antibody are V_(H)CDR1.4, V_(H)CDR2.4 and V_(H)CDR3.4. In aspecific embodiment, the V_(H) chain of the anti-hPG monoclonal antibodyhas a sequence corresponding to mV_(H).4 (SEQ ID NO:14). See FIG. 2C.

In some embodiments, the CDRs of the anti-hPG monoclonal antibody V_(L)chain are V_(L)CDR1.4, V_(L)CDR2.4 and V_(L)CDR3.4 In a specificembodiment, the V_(L) chain of the anti-hPG monoclonal antibody has anamino acid sequence corresponding to mV_(L).4 (SEQ ID NO:15). See FIG.2D.

In some embodiments, the CDRs of the V_(H) chain of the anti-hPGmonoclonal antibody are V_(H)CDR1.8, V_(H)CDR2.8 and V_(H)CDR3.8. In aspecific embodiment, the V_(H) chain of the anti-hPG monoclonal antibodyhas a sequence corresponding to mV_(H).8 (SEQ ID NO:59). See FIG. 2E.

In some embodiments, the CDRs of the anti-hPG monoclonal antibody V_(L)chain are V_(L) CDR1.8, V_(L) CDR2.8 and V_(L) CDR3.8. In a specificembodiment, the V_(L) chain of the anti-hPG monoclonal antibody has anamino acid sequence corresponding to mV_(L).8 (SEQ ID NO:63). See FIG.2F.

In some embodiments, the CDRs of the V_(H) chain of the anti-hPGmonoclonal antibody are V_(H)CDR1.13, V_(H)CDR2.13 and V_(H)CDR3.13. Ina specific embodiment, the V_(H) chain of the anti-hPG monoclonalantibody has a sequence corresponding to mV_(H).13 (SEQ ID NO:60). SeeFIG. 2G.

In some embodiments, the CDRs of the anti-hPG monoclonal antibody V_(L)chain are V_(L) CDR1.13, V_(L) CDR2.13 and V_(L) CDR3.13. In a specificembodiment, the V_(L) chain of the anti-hPG monoclonal antibody has anamino acid sequence corresponding to mV_(L).13 (SEQ ID NO:64). See FIG.2H.

In some embodiments, the CDRs of the V_(H) chain of the anti-hPGmonoclonal antibody are V_(H)CDR1.16, V_(H)CDR2.16 and V_(H)CDR3.16. Ina specific embodiment, the V_(H) chain of the anti-hPG monoclonalantibody has a sequence corresponding to mV_(H).16 (SEQ ID NO:61). SeeFIG. 2I.

In some embodiments, the CDRs of the anti-hPG monoclonal antibody V_(L)chain are V_(L) CDR1.16, V_(L) CDR2.16 and V_(L) CDR3.16. In a specificembodiment, the V_(L) chain of the anti-hPG monoclonal antibody has anamino acid sequence corresponding to mV_(L).16 (SEQ ID NO:65). See FIG.2J.

In some embodiments, the CDRs of the V_(H) chain of the anti-hPGmonoclonal antibody are V_(H)CDR1.19, V_(H)CDR2.19 and V_(H)CDR3.19. Ina specific embodiment, the V_(H) chain of the anti-hPG monoclonalantibody has a sequence corresponding to mV_(H).19 (SEQ ID NO:62). SeeFIG. 2K.

In some embodiments, the CDRs of the anti-hPG monoclonal antibody V_(L)chain are V_(L) CDR1.19, V_(L) CDR2.19 and V_(L) CDR3.19. In a specificembodiment, the V_(L) chain of the anti-hPG monoclonal antibody has anamino acid sequence corresponding to mV_(L).19 (SEQ ID NO:66). See FIG.2L.

In some embodiments, the CDRs of the V_(H) chain of the anti-hPGmonoclonal antibody are V_(H)CDR1.3, V_(H)CDR2.3 and V_(H)CDR3.3 and theCDRs of the V_(L) chain are V_(L)CDR1.3, V_(L)CDR2.3 and V_(L)CDR3.3. Ina specific embodiment, the V_(H) chain of the anti-PG monoclonalantibody has an amino acid sequence corresponding to mV_(H).3 (SEQ IDNO: 12) and the V_(L) chain has a sequence corresponding to mV_(L).3(SEQ ID NO:13).

In some embodiments, the CDRs of the V_(H) chain of the anti-hPGmonoclonal antibody are V_(H)CDR1.4, V_(H)CDR2.4 and V_(H)CDR3.4 and theCDRs of the V_(L) chain are V_(L)CDR1.4, V_(L)CDR2.4 and V_(L)CDR3.4. Ina specific embodiment, the V_(H) chain of the anti-hPG monoclonalantibody has an amino acid sequence corresponding to mV_(H).4 (SEQ IDNO:14) and a V_(L) chain has an amino acid sequence corresponding tomV_(L).4 (SEQ ID NO:15).

In some embodiments, the CDRs of the V_(H) chain of the anti-hPGmonoclonal antibody are V_(H)CDR1.8, V_(H)CDR2.8 and V_(H)CDR3.8 and theCDRs of the V_(L) chain are V_(L)CDR1.8, V_(L)CDR2.8 and V_(L)CDR3.8. Ina specific embodiment, the anti-hPG monoclonal antibody is anti-hPG MAb8 described herein and comprises an amino acid sequence corresponding tomV_(H).8 (SEQ ID NO:59) and an amino acid sequence corresponding tomV_(L).8 (SEQ ID NO:63).

In some embodiments, the CDRs of the V_(H) chain of the anti-hPGmonoclonal antibody are V_(H)CDR1.3, V_(H)CDR2.13 and V_(H)CDR3.13 andthe CDRs of the V_(L) chain are V_(L)CDR1.13, V_(L)CDR2.13 andV_(L)CDR3.13. In a specific embodiment, the anti-hPG monoclonal antibodyis anti-hPG MAb 13 described herein and comprises an amino acid sequencecorresponding to mV_(H).13 (SEQ ID NO:60) and an amino acid sequencecorresponding to mV_(L).13 (SEQ ID NO:64).

In some embodiments, the CDRs of the V_(H) chain of the anti-hPGmonoclonal antibody are V_(H)CDR1.16, V_(H)CDR2.16 and V_(H)CDR3.16 andthe CDRs of the V_(L) chain are V_(L)CDR1.16, V_(L)CDR2.16 andV_(L)CDR3.16. In a specific embodiment, the anti-hPG monoclonal antibodyis anti-hPG MAb 16 described herein and comprises an amino acid sequencecorresponding to mV_(H).16 (SEQ ID NO:61) and an amino acid sequencecorresponding to mV_(L).16 (SEQ ID NO:65).

In some embodiments, the CDRs of the V_(H) chain of the anti-hPGmonoclonal antibody are V_(H)CDR1.19, V_(H)CDR2.19 and V_(H)CDR3.19 andthe CDRs of the V_(L) chain are V_(L)CDR1.19, V_(L)CDR2.19 andV_(L)CDR3.19. In a specific embodiment, the anti-hPG monoclonal antibodyis anti-hPG MAb 19 described herein and comprises an amino acid sequencecorresponding to mV_(H).19 (SEQ ID NO:62) and an amino acid sequencecorresponding to mV_(L).19 (SEQ ID NO:66).

Anti-hPG monoclonal antibodies of the disclosure include both intactmolecules, and antibody fragments (such as, for example, Fab and F(ab′)₂fragments) which are capable of specifically binding to hPG. Fab andF(ab′)₂ fragments lack the Fc fragment of intact antibody, clear morerapidly from the circulation of the animal or plant, and may have lessnon-specific tissue binding than an intact antibody (Wahl et al., 1983,J. Nucl. Med. 24:316). Antibody fragments are therefore useful intherapeutic applications among other applications.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the target binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)2 and Fv fragments. An “Fv”fragment is the minimum antibody fragment which contains a completetarget recognition and binding site. This region consists of a dimer ofone heavy and one light chain variable domain in a tight, non-covalentassociation (V_(H)-V_(L) dimer). It is in this configuration that thethree CDRs of each variable domain interact to define a target bindingsite on the surface of the V_(H)-V_(L) dimer. Often, the six CDRs confertarget binding specificity to the antibody. However, in some instanceseven a single variable domain (or half of an Fv comprising only threeCDRs specific for a target) can have the ability to recognize and bindtarget, although at a lower affinity than the entire binding site.“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of an antibody, wherein these domains are present in asingle polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the scFv to form the desired structure for target binding.“Single domain antibodies” are composed of a single V_(H) or V_(L)domains which exhibit sufficient affinity to hPG. In a specificembodiment, the single domain antibody is a camelized antibody (See,e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38).

The Fab fragment contains the constant domain of the light chain and thefirst constant domain (CH₁) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH₁ domain including one or more cysteinesfrom the antibody hinge region. F(ab′) fragments are produced bycleavage of the disulfide bond at the hinge cysteines of the F(ab′)₂pepsin digestion product. Additional chemical couplings of antibodyfragments are known to those of ordinary skill in the art.

The anti-hPG monoclonal antibodies of the disclosure can be chimericantibodies. The term “chimeric” antibody as used herein refers to anantibody having variable sequences derived from a non-humanimmunoglobulins, such as rat or mouse antibody, and humanimmunoglobulins constant regions, typically chosen from a humanimmunoglobulin template. Methods for producing chimeric antibodies areknown in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7;Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J.Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and4,816,397, which are incorporated herein by reference in theirentireties.

The anti-hPG monoclonal antibodies of the disclosure can be humanized.“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other target-binding subsequences of antibodies)which contain minimal sequences derived from non-human immunoglobulins.In general, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin consensus sequence, and can be referred to as“CDR-grafted.” The humanized antibody can also comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin consensus sequence. Methods of antibodyhumanization, including methods of designing humanized antibodies, areknown in the art. See, e.g., Lefranc et al., 2003, Dev. Comp. Immunol.27:55-77; Lefranc et al., 2009, Nucl. Acids Res. 37: D1006-1012;Lefranc, 2008, Mol. Biotechnol. 40: 101-111; Riechmann et al., 1988,Nature 332:323-7; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761;5,693,762; and 6,180,370 to Queen et al.; EP239400; PCT publication WO91/09967; U.S. Pat. No. 5,225,539; EP592106; EP519596; Padlan, 1991,Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814;Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Pat.No. 5,565,332, all of which are hereby incorporated by reference intheir entireties.

Sequences for humanized anti-hPG monoclonal antibodies were designedfrom murine anti-hPG monoclonal antibodies of the present disclosure asdescribed in the Examples below. Specific embodiments of humanizedantibodies include antibodies comprising: (1) any three VL CDRs and anythree VH CDRs disclosed herein; (2) a heavy chain variable regioncomprising an amino acid sequence corresponding to SEQ ID NO:21 and alight chain variable region comprising an amino acid sequencecorresponding to SEQ ID NO:22; (3) a heavy chain variable regioncomprising an amino acid sequence corresponding to SEQ ID NO:23 and alight chain variable region comprising an amino acid sequencecorresponding to SEQ ID NO:24; (4) a heavy chain variable regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:75, 77, and 79 and a light chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NO:76and 78; (5) a heavy chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NO:80 and 82 and alight chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:81 and 83; (6) a heavy chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:84, 86, and 88 and a light chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:85, 87, and 89; (7) a heavy chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:90, 92, and 94 and a light chain variable regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:91, 93, and 95.

The anti-hPG monoclonal antibodies of the disclosure can be primatized.The term “primatized antibody” refers to an antibody comprising monkeyvariable regions and human constant regions. Methods for producingprimatized antibodies are known in the art. See, e.g., U.S. Pat. Nos.5,658,570; 5,681,722; and 5,693,780, which are incorporated herein byreference in their entireties.

Included within anti-hPG monoclonal antibodies are antibodies thatcompete with a reference antibody, such as, for example, a polyclonalanti-hPG antibody, or any of the anti-hPG monoclonal antibodiesdisclosed herein. Antibodies that compete with the anti-hPG monoclonalantibodies of the disclosure are useful in for a variety of diagnosticand therapeutic applications. Specific embodiments of suitable referenceanti-hPG monoclonal antibodies include the antibodies described herein,for example, but not by way of limitation: antibodies comprising anythree V_(L) CDRs and any three V_(H) CDRs disclosed herein; antibodiesin which the V_(H) chain has an amino acid sequence corresponding to SEQID NO:12 (mV_(H).3) and the V_(L) chain has an amino acid sequencecorresponding to SEQ ID NO:13 (mV_(L).3); and antibodies in which theV_(H) chain has an amino acid sequence corresponding to SEQ ID NO:14(mV_(H).4) and the V_(L) chain has a sequence corresponding to SEQ IDNO:15 (mV_(L).4), antibodies in which the VH chain has an amino acidsequence corresponding to SEQ ID NO:59 (mV_(H).8) and the VL chain hasan amino acid sequence corresponding to SEQ ID NO:63 (mV_(L).8);antibodies in which the V_(H) chain has an amino acid sequencecorresponding to SEQ ID NO:60 (mV_(H).13) and the V_(L) chain has anamino acid sequence corresponding to SEQ ID NO:64 (mV_(L).13);antibodies in which the V_(H) chain has an amino acid sequencecorresponding to SEQ ID NO:61 (mV_(H).16) and the V_(L) chain has anamino acid sequence corresponding to SEQ ID NO:65 (mV_(L).16);antibodies in which the V_(H) chain has an amino acid sequencecorresponding to SEQ ID NO:62 (mV_(H).19) and the V_(L) chain has anamino acid sequence corresponding to SEQ ID NO:66 (mV_(L).19) or anycombination of V_(H) and V_(L) chains disclosed herein.

Suitable reference antibodies also include antibodies produced by ahybridoma selected from the group consisting of 43B9G11, WE5H2G7,6B5B11C10, 20D2C3G2, 1B4A11D11, 1B6A11F2, 1B11E4B11, 1C10D3B9, 1D8F5B3,1E1C7B4, 2B4C8C8, 2B11E6G4, 2C6C3C7, 2H9F4B7, 1E9A4A4, 1E9D9B6,1C8D10F5, 1A7C3F11, 1B3B4F11, 1C11F5E8, 1F11F5E10, 1F11F5G9, and1A11F2C9; antibodies that bind an epitope that includes residues 10 to14 of hPG (SEQ ID NO:28), residues 9 to 14 of hPG (SEQ ID NO:29),residues 4 to 10 of hPG (SEQ ID NO:30), residues 2 to 10 of hPG (SEQ IDNO:31), or residues 2 to 14 of hPG (SEQ ID NO:32); and antibodies thatbind an epitope that includes residues 71 to 74 of hPG (SEQ ID NO:33),residues 69 to 73 of hPG (SEQ ID NO:34), residues 76 to 80 of hPG (SEQID NO:35), or residues 67 to 74 of hPG (SEQ ID NO:36).

The ability to compete with a monoclonal antibody of the presentdisclosure for binding to PG, for example hPG, can be tested using acompetition assay as follows. 96-well plates are coated with a captureantibody (polyclonal or monoclonal antibody recognizing an N- orC-terminal region of progastrin that differs from the epitope recognizedby the reference monoclonal antibody), at a concentration to be chosenwithin the range of 1-10 μg/ml, overnight at 4° C. (0.1 to 1 μg/well).After blocking with 0.1% Tween-20/0.1% BSA in PBS (blocking buffer) for2 h at 22° C., recombinant human progastrin is added at a concentrationof 10 pM to 1 nM (10 to 1000 pg/well) and incubated for 2 h at 22° C.Thereafter, the biotinylated reference anti-hPG monoclonal antibody or amixture containing the reference monoclonal antibody is added along withincreasing concentrations of unlabeled test antibodies and incubated for1 hour at 22° C. After washing, detection is performed by incubating for1 h at 22° C. with a fluorogenic substrate for horseradish peroxidase,followed by quantitation of relative light units (RLU) in a luminometer.Assays are performed in duplicate. Antibodies that compete with areference anti-hPG monoclonal antibody of the present disclosure inhibitthe binding of the reference antibody to hPG. An antibody that binds tothe same epitope as the control antibodies will be able to effectivelycompete for binding and thus will significantly reduce (for example, byat least 50%) the reference antibody binding, as evidenced by areduction in the bound label. The reactivity of the (labeled) referenceantibodies in the absence of a completely irrelevant antibody would bethe control high value. The control low value would be obtained byincubating the unlabeled test antibodies with cells expressingprogastrin and then incubate the cell/antibody mixture with labeledcontrol antibodies of exactly the same type, when competition wouldoccur and reduce binding of the labeled antibodies. In a test assay, asignificant reduction in labeled antibody reactivity in the presence ofa test antibody is indicative of a test antibody that recognizessubstantially the same epitope.

The binding inhibition can be expressed as an inhibition constant, orKi, which is calculated according to the following formula:

Ki=IC50/(1+[Reference Ab concentration]/Kd)

Where IC50 of the test antibody is the concentration of test antibodythat yields a 50% reduction in binding of the reference antibody and Kdis the dissociation constant of the reference antibody, a measure of itsaffinity for progastrin. Antibodies that compete with anti-hPGmonoclonal antibodies disclosed herein can have a Ki from 10 pM to 10 nMunder assay conditions described herein.

In various embodiments, an unlabeled anti-hPG monoclonal antibody of thedisclosure reduces the binding of labeled reference antibody by at least40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%,by at least 90%, by 100%, or by a percentage ranging between any of theforegoing values (e.g., an anti-hPG monoclonal antibody of thedisclosure reduces the binding of labeled reference antibody by 50% to70%) when the anti-hPG monoclonal antibody is used at a concentration of0.01 μg/ml, 0.08 μg/ml, 0.4 μg/ml, 2 μg/ml, 10 μg/ml, 50 μg/ml, 100μg/ml or at a concentration ranging between any of the foregoing values(e.g., at a concentration ranging from 2 μg/ml to 10 μg/ml).

In conducting an antibody competition study between a reference antibodyand any test antibody (irrespective of species or isotype), one mayfirst label the reference with a detectable label, such as, biotin or anenzymatic (or even radioactive) label to enable subsequentidentification. In this case, labeled reference antibody (in fixed orincreasing concentrations) is incubated with a known amount ofprogastrin. The unlabeled test antibody is then added to the pre-boundcomplex of progastrin and labeled antibody. The intensity of the boundlabel is measured. If the test antibody competes with the labeledantibody by binding to an overlapping epitope, the intensity will bedecreased relative to the binding of the control labeled antibody in theabsence of the test antibody.

Assays for competition are known and can be adapted to yield comparableresults to the assay described above. The assay may be any one of arange of immunological assays based upon antibody competition, and thereference antibodies would be detected by means of detecting theirlabel, e.g., by using streptavidin in the case of biotinylatedantibodies or by using a chromogenic substrate in connection with anenzymatic label (such as 3,3′5,5′-tetramethylbenzidine (TMB) substratewith peroxidase enzyme) or by simply detecting a radioactive label or afluorescence label.

Also included herein are anti-hPG monoclonal antibodies which arederivatized, covalently modified, or conjugated to other molecules, foruse in diagnostic and therapeutic applications. For example, but not byway of limitation, derivatized antibodies include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications can be carried outby known techniques, including, but not limited to, specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative can contain one or more non-classicalamino acids.

In another example, antibodies of the present disclosure can be attachedto poly(ethyleneglycol) (PEG) moieties. In a specific embodiment, theantibody is an antibody fragment and the PEG moieties are attachedthrough any available amino acid side-chain or terminal amino acidfunctional group located in the antibody fragment, for example any freeamino, imino, thiol, hydroxyl or carboxyl group. Such amino acids canoccur naturally in the antibody fragment or can be engineered into thefragment using recombinant DNA methods. See, for example U.S. Pat. No.5,219,996. Multiple sites can be used to attach two or more PEGmolecules. PEG moieties can be covalently linked through a thiol groupof at least one cysteine residue located in the antibody fragment. Wherea thiol group is used as the point of attachment, appropriatelyactivated effector moieties, for example thiol selective derivativessuch as maleimides and cysteine derivatives, can be used.

In a specific example, an anti-hPG monoclonal antibody conjugate is amodified Fab′ fragment which is PEGylated, i.e., has PEG(poly(ethyleneglycol)) covalently attached thereto, e.g., according tothe method disclosed in EP0948544. See also Poly(ethyleneglycol)Chemistry, Biotechnical and Biomedical Applications, (J. Milton Harris(ed.), Plenum Press, New York, 1992); Poly(ethyleneglycol) Chemistry andBiological Applications, (J. Milton Harris and S. Zalipsky, eds.,American Chemical Society, Washington D.C., 1997); and BioconjugationProtein Coupling Techniques for the Biomedical Sciences, (M. Aslam andA. Dent, eds., Grove Publishers, New York, 1998); and Chapman, 2002,Advanced Drug Delivery Reviews 54:531-545. PEG can be attached to acysteine in the hinge region. In one example, a PEG-modified Fab′fragment has a maleimide group covalently linked to a single thiol groupin a modified hinge region. A lysine residue can be covalently linked tothe maleimide group and to each of the amine groups on the lysineresidue can be attached a methoxypoly(ethyleneglycol) polymer having amolecular weight of approximately 20,000 Da. The total molecular weightof the PEG attached to the Fab′ fragment can therefore be approximately40,000 Da.

Anti-hPG monoclonal antibodies include labeled antibodies, useful indiagnostic applications. The antibodies can be used diagnostically, forexample, to detect expression of a target of interest in specific cells,tissues, or serum; or to monitor the development or progression of animmunologic response as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance or“label.” A label can be conjugated directly or indirectly to an anti-hPGmonoclonal antibody of the disclosure. The label can itself bedetectable (e.g., radioisotope labels, isotopic labels, or fluorescentlabels) or, in the case of an enzymatic label, can catalyze chemicalalteration of a substrate compound or composition which is detectable.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, radioactive materials, positron emitting metals using variouspositron emission tomographies, and nonradioactive paramagnetic metalions. The detectable substance can be coupled or conjugated eitherdirectly to the antibody (or fragment thereof) or indirectly, through anintermediate (such as, for example, a linker known in the art) usingtechniques known in the art. Examples of enzymatic labels includeluciferases (e.g., firefly luciferase and bacterial luciferase; U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malatedehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO),alkaline phosphatase, β-galactosidase, acetylcholinesterase,glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclicoxidases (such as uricase and xanthine oxidase), lactoperoxidase,microperoxidase, and the like. Examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin; examples ofsuitable fluorescent materials include umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride, dimethylamine-1-napthalenesulfonylchloride, or phycoerythrin and the like; an example of a luminescentmaterial includes luminol; examples of bioluminescent materials includeluciferase, luciferin, and aequorin; examples of suitable isotopicmaterials include ¹³C, ¹⁵N, and deuterium; and examples of suitableradioactive material include ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

Anti-hPG monoclonal antibodies of all species of origin are included inthe present invention. Non-limiting exemplary natural antibodies includeantibodies derived from humans, simians, chicken, goats, rabbits, androdents (e.g., rats, mice, and hamsters) (See, e.g., Lonberg et al.,WO93/12227; U.S. Pat. No. 5,545,806; and Kucherlapati, et al.,WO91/10741; U.S. Pat. No. 6,150,584, which are herein incorporated byreference in their entirety). Natural antibodies are the antibodiesproduced by a host animal after being immunized with an antigen, such asa polypeptide.

Nucleic Acids and Expression Systems

The present disclosure encompasses nucleic acid molecules encodingimmunoglobulin light and heavy chain genes for anti-hPG monoclonalantibodies, vectors comprising such nucleic acids, and host cellscapable of producing the anti-hPG monoclonal antibodies of thedisclosure.

An anti-hPG monoclonal antibody of the disclosure can be prepared byrecombinant expression of immunoglobulin light and heavy chain genes ina host cell. To express an antibody recombinantly, a host cell istransfected with one or more recombinant expression vectors carrying DNAfragments encoding the immunoglobulin light and heavy chains of theantibody such that the light and heavy chains are expressed in the hostcell and, optionally, secreted into the medium in which the host cellsare cultured, from which medium the antibodies can be recovered.Standard recombinant DNA methodologies are used to obtain antibody heavyand light chain genes, incorporate these genes into recombinantexpression vectors and introduce the vectors into host cells, such asthose described in Molecular Cloning; A Laboratory Manual, SecondEdition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N.Y.,1989), Current Protocols in Molecular Biology (Ausubel, F. M. et al.,eds., Greene Publishing Associates, 1989) and in U.S. Pat. No.4,816,397.

To generate nucleic acids encoding such anti-hPG monoclonal antibodies,DNA fragments encoding the light and heavy chain variable regions arefirst obtained. These DNAs can be obtained by amplification andmodification of germline DNA or cDNA encoding light and heavy chainvariable sequences, for example using the polymerase chain reaction(PCR). Germline DNA sequences for human heavy and light chain variableregion genes are known in the art (See, e.g., Lefranc et al., 2003, Dev.Comp. Immunol. 27:55-77; Lefranc et al., 2009, Nucl. Acids Res. 37:D1006-1012; Lefranc, 2008, Mol. Biotechnol. 40: 101-111; the “VBASE”human germline sequence database; see also Kabat, E. A. et al., 1991,Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242;Tomlinson et al., 1992, J. Mol. Biol. 22T:116-198; and Cox et al., 1994,Eur. J. Immunol. 24:827-836; the contents of each of which areincorporated herein by reference).

Once DNA fragments encoding anti-hPG monoclonal antibody-related V_(H)and V_(L) segments are obtained, these DNA fragments can be furthermanipulated by standard recombinant DNA techniques, for example toconvert the variable region genes to full-length antibody chain genes,to Fab fragment genes or to a scFv gene. In these manipulations, aV_(L)- or V_(H)-encoding DNA fragment is operatively linked to anotherDNA fragment encoding another protein, such as an antibody constantregion or a flexible linker. The term “operatively linked,” as used inthis context, is intended to mean that the two DNA fragments are joinedsuch that the amino acid sequences encoded by the two DNA fragmentsremain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH₁,CH₂, CH₃ and, optionally, CH₄). The sequences of human heavy chainconstant region genes are known in the art (See, e.g., Kabat, E. A., etal., 1991, Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The heavy chain constant regioncan be an IgG₁, IgG₂, IgG₃, IgG₄, IgA, IgE, IgM or IgD constant region,but in certain embodiments is an IgG₁ or IgG₄ constant region. For a Fabfragment heavy chain gene, the V_(H)-encoding DNA can be operativelylinked to another DNA molecule encoding only the heavy chain CH₁constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (See, e.g.,Kabat, E. A., et al., 1991, Sequences of Proteins of ImmunologicalInterest, Fifth Edition (U.S. Department of Health and Human Services,NIH Publication No. 91-3242)) and DNA fragments encompassing theseregions can be obtained by standard PCR amplification. The light chainconstant region can be a kappa or lambda constant region, but in certainembodiments is a kappa constant region. To create a scFv gene, theV_(H)- and V_(L)-encoding DNA fragments are operatively linked toanother fragment encoding a flexible linker, e.g., encoding the aminoacid sequence (Gly₄˜Ser)₃ (SEQ ID NO: 99), such that the V_(H) and V_(L)sequences can be expressed as a contiguous single-chain protein, withthe V_(L) and V_(H) regions joined by the flexible linker (See, e.g.,Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature348:552-554).

To express the anti-hPG monoclonal antibodies of the disclosure, DNAsencoding partial or full-length light and heavy chains, obtained asdescribed above, are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalcontrol sequences. In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vectors or, more typically, bothgenes are inserted into the same expression vector.

The antibody genes are inserted into the expression vector by standardmethods (e.g., ligation of complementary restriction sites on theantibody gene fragment and vector, or blunt end ligation if norestriction sites are present). Prior to insertion of the anti-hPGmonoclonal antibody-related light or heavy chain sequences, theexpression vector can already carry antibody constant region sequences.For example, one approach to converting the anti-hPG monoclonalantibody-related V_(H) and V_(L) sequences to full-length antibody genesis to insert them into expression vectors already encoding heavy chainconstant and light chain constant regions, respectively, such that theV_(H) segment is operatively linked to the CH segment(s) within thevector and the V_(L) segment is operatively linked to the CL segmentwithin the vector. Additionally or alternatively, the recombinantexpression vector can encode a signal peptide that facilitates secretionof the antibody chain from a host cell. The antibody chain gene can becloned into the vector such that the signal peptide is linked in-frameto the amino terminus of the antibody chain gene. The signal peptide canbe an immunoglobulin signal peptide or a heterologous signal peptide(i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the disclosure carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel, GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif., 1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Suitable regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, see,e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al., and U.S. Pat. No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the disclosure can carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (See, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Suitable selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in DHFR⁻ host cells withmethotrexate selection/amplification) and the neo gene (for G418selection). For expression of the light and heavy chains, the expressionvector(s) encoding the heavy and light chains is transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, lipofection,calcium-phosphate precipitation, DEAE-dextran transfection and the like.

It is possible to express the antibodies of the disclosure in eitherprokaryotic or eukaryotic host cells. In certain embodiments, expressionof antibodies is performed in eukaryotic cells, e.g., mammalian hostcells, of optimal secretion of a properly folded and immunologicallyactive antibody. Exemplary mammalian host cells for expressing therecombinant antibodies of the disclosure include Chinese Hamster Ovary(CHO cells) (including DHFR⁻ CHO cells, described in Urlaub and Chasin,1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFRselectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol.Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods. Host cells can also be used to produce portions of intactantibodies, such as Fab fragments or scFv molecules. It is understoodthat variations on the above procedure are within the scope of thepresent disclosure. For example, it can be desirable to transfect a hostcell with DNA encoding either the light chain or the heavy chain (butnot both) of an anti-hPG monoclonal antibody of this disclosure.

Recombinant DNA technology can also be used to remove some or all of theDNA encoding either or both of the light and heavy chains that is notnecessary for binding to hPG. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of thedisclosure.

For recombinant expression of an anti-hPG monoclonal antibody of thedisclosure, the host cell can be co-transfected with two expressionvectors of the disclosure, the first vector encoding a heavy chainderived polypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors can contain identical selectable markers,or they can each contain a separate selectable marker. Alternatively, asingle vector can be used which encodes both heavy and light chainpolypeptides.

Once a nucleic acid encoding one or more portions of an anti-hPGmonoclonal antibody, further alterations or mutations can be introducedinto the coding sequence, for example to generate nucleic acids encodingantibodies with different CDR sequences, antibodies with reducedaffinity to the Fc receptor, or antibodies of different subclasses.

The anti-hPG monoclonal antibodies of the disclosure can also beproduced by chemical synthesis (e.g., by the methods described in SolidPhase Peptide Synthesis, 2^(nd) ed., 1984 The Pierce Chemical Co.,Rockford, Ill.). Variant antibodies can also be generated using acell-free platform (See, e.g., Chu et al., Biochemia No. 2, 2001 (RocheMolecular Biologicals).

Once an anti-hPG monoclonal antibody of the disclosure has been producedby recombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theanti-hPG monoclonal antibodies of the present disclosure or fragmentsthereof can be fused to heterologous polypeptide sequences describedherein or otherwise known in the art to facilitate purification.

Once isolated, the anti-hPG monoclonal antibody can, if desired, befurther purified, e.g., by high performance liquid chromatography (See,e.g., Fisher, Laboratory Techniques In Biochemistry And MolecularBiology (Work and Burdon, eds., Elsevier, 1980), or by gel filtrationchromatography on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala,Sweden).

The present disclosure provides host cells capable of producing anti-hPGmonoclonal antibodies. Host cells can be cells engineered usingrecombinant DNA techniques to express genes encoding heavy and lightchain genes or hybridomas derived from a suitable organism and selectedfor the ability to produce the desired antibodies.

Host cells capable of producing anti-PG monoclonal antibodies can behybridomas. Methods for generating hybridomas are known in the art (see,e.g., Kohler and Milstein, 1975, Nature 256:495) and an example isprovided below. Generally, a host animal, such as a mouse is immunizedwith an immunogen, such as a peptide of interest, to elicit thedevelopment of lymphocytes, for example spleen cells, that produceantibodies capable of specifically binding the immunogen. Alternatively,isolated lymphocytes, including spleen cells, lymph node cells, orperipheral blood lymphocytes, can be immunized in vitro. Lymphocytes arethen fused with an immortalized cell line, such as a myeloma cell line,using a suitable fusing agent (e.g., polyethylene glycol), to form ahybridoma cell line. Suitable immortalized cell lines can be ofmammalian origin, such as murine, bovine, or human. Hybridoma cells arethen cultured in any suitable medium that contains one or moresubstances that inhibit growth or survival of the unfused, immortalizedcells. For example, when parental cells lacking the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT) are used, fusions canbe grown in medium containing hypoxanthine, aminopterin, and thymidine(“HAT” medium) which inhibits the growth of parental, unfused cells.

In some embodiments, the N-terminal anti-hPG monoclonal antibodies havevariable light chain (V_(L)) CDRs that correspond to the V_(L) of themonoclonal antibody obtainable from hybridomas 43B9G11 (producinganti-hPG MAb 1), WE5H2G7 (producing anti-hPG MAb 2), 6B5B11C10(producing anti-hPG MAb 3), 20D2C3G2 (producing anti-hPG MAb 4), 1E9A4A4 (producing anti-hPG MAb 15), 1 E9D9B6 (producing anti-hPG MAb16), 1C8D10F5 (producing anti-hPG MAb 17), 1A7C3F11 (producing anti-hPGMAb 18), 1B3B4F11 (producing anti-hPG MAb 19), and 1C11F5E8 (producinganti-hPG MAb 20).

In some embodiments, the N-terminal anti-hPG monoclonal antibodies haveV_(H) CDRs that correspond to the V_(H) CDRs of the monoclonalantibodies obtainable from the above hybridomas.

In some embodiments, the C-terminal anti-hPG monoclonal antibodies haveV_(L) CDRs that correspond to the V_(L) of the monoclonal antibodyobtainable from hybridomas 1B4A11D11 (producing anti-hPG MAb 5),1B6A11F2 (producing anti-hPG MAb 6), 1B11E4B11 (producing anti-hPG MAb7), 1C10D3B9 (producing anti-hPG MAb 8), 1D8F5B3 (producing anti-hPG MAb9), 1E1C7B4 (producing anti-hPG MAb 10), 2B4C8C8 (producing anti-hPG MAb11), 2B11E6G4 (producing anti-hPG MAb 12), 2C6C3C7 (producing anti-hPGMAb 13), 2H9F4B7 (producing anti-hPG MAb 14), 1F11F5E10 (producinganti-hPG MAb 21), 1F11F5G9 (producing anti-hPG MAb 22), and 1A11F2C9(producing anti-hPG MAb 23).

In some embodiments, the C-terminal anti-hPG monoclonal antibodies haveV_(H) CDRs that correspond to the V_(H) CDRs of the monoclonalantibodies obtainable from the above hybridomas.

In an embodiment, a host cell capable of producing an anti-hPG antibodycomprising a heavy chain variable region comprising SEQ ID NO:12 and alight chain variable region comprising SEQ ID NO:13 is provided. In anembodiment, a host cell capable of producing an anti-hPG antibodycomprising a heavy chain variable region comprising SEQ ID NO:14 and alight chain variable region comprising SEQ ID NO:15 is provided. In anembodiment, a host cell capable of producing an anti-hPG antibodycomprising a heavy chain variable region comprising SEQ ID NO:59 and alight chain variable region comprising SEQ ID NO:63 is provided. In anembodiment, a host cell capable of producing an anti-hPG antibodycomprising a heavy chain variable region comprising SEQ ID NO:60 and alight chain variable region comprising SEQ ID NO:64 is provided. In anembodiment, a host cell capable of producing an anti-hPG antibodycomprising a heavy chain variable region comprising SEQ ID NO:61 and alight chain variable region comprising SEQ ID NO:65 is provided. In anembodiment, a host cell capable of producing an anti-hPG antibodycomprising a heavy chain variable region comprising SEQ ID NO:62 and alight chain variable region comprising SEQ ID NO:66 is provided.

In some embodiments, a host cell of the disclosure comprises a nucleicacid selected from: a nucleotide sequence encoding the heavy chainvariable region polypeptide of SEQ ID NOs:12, 14, 59, 60, 61, and 62;and a nucleic acid selected from: a nucleotide sequence encoding thelight chain variable region polypeptide of SEQ ID NOs:13, 15, 63, 64,65, and 66. In some embodiments, the heavy chain variable region isencoded by a nucleic acid sequence selected from: SEQ ID NOs:16, 18, 67,68, 69 and 70. In some embodiments, the light chain variable region isencoded by a nucleic acid sequence selected from: SEQ ID NOs:17, 19, 71,72, 73, and 74.

In some embodiments, polynucleotide sequences are provided encoding aheavy chain variable region of a humanized anti-hPG monoclonal antibody.Specific embodiments include polynucleotides encoding a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:21, 23, 75, 77, 79, 80, 82, 84, 86, 88, 90, 92, and 94. In someembodiments, polynucleotide sequences are provided encoding a lightchain variable region of a humanized anti-hPG monoclonal antibody.Specific embodiments include polynucleotides encoding a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:22, 24, 76, 78, 81, 83, 85, 87, 89, 91, 93, and 95.

Biological Activities of Anti-hPG Monoclonal Antibodies

PG has been implicated in CRC tumor cell survival and/or proliferation.As described above in the Detailed Description, neutralizing anti-PGmonoclonal antibodies are thought to block or inhibit PG's ability tointeract with its signaling partner(s). As a consequence of theseactivities, neutralizing anti-hPG monoclonal antibodies of thedisclosure can be used in a variety of in vitro, in vivo, and ex vivocontexts to bind PG and block PG-dependent signaling.

Accordingly, the disclosure provides methods of inhibiting PG-dependentresponses in CRC cells. Generally, the methods comprise contacting a CRCcell with, or exposing a cell population to, a neutralizing anti-PGmonoclonal antibody in an amount effective to inhibit one or morePG-induced responses of CRC cells, e.g. proliferation and/or survival ofCRC cells. Proliferation, or inhibition thereof, in vitro and in vivocan be determined according to assays for measuring increases in cellnumber, tumor number, or tumor size over time. Assays for inhibition ofproliferation of cells and tumors are well known in the art.

Blocking PG-dependent signaling can inhibit survival of CRC cells byincreasing cell death. Inhibition of CRC cell survival in vitro or invivo can be determined by measuring reduction of live cancer cellnumbers over time (e.g., 24 or 48 hours). Assays for cell death are wellknown in the art. Additionally, an example of a cell survival assay isprovided herein.

Studies further suggest that inhibiting PG-dependent signaling in CRCtumor cells can inhibit survival of CRC cells by trigger programmed celldeath, or apoptosis. Induction of apoptosis can be determined by anymeans known in the art, including but not limited to, measuring changesin expression of genes having pro- or anti-apoptotic activity. Forexample, an increase in expression over time (e.g., 48 hours) of apro-apoptotic gene, such as, for example, Bax, is indicative of anincrease in apoptosis. Similarly, a decrease in expression over time(e.g., 72 or 96 hours) of an anti-apoptotic gene, such as, for example,but not by way of limitation, Bcl-2, is indicative of an increase inapoptosis. Techniques for measuring changes in gene expression, such asreal-time quantitative PCR, are well known in the art. See, e.g.,Hollande et al., WO 2007/135542.

Inhibition of progastrin-dependent signaling also stimulates celldifferentiation. Accordingly, methods of inhibiting proliferation and/orsurvival of CRC cells comprise administering an amount of a neutralizinganti-PG monoclonal antibody effective to induce differentiation of CRCcells in vitro or in vivo. Differentiation of CRC cells can bedetermined by measuring increases over time (e.g., 24 or 48 hours) inexpression of genetic markers for cellular differentiation, such as, forexample, but not by way of limitation, Muc-2 or other markers fordifferentiated intestinal cells (e.g., goblet cells). Changes in geneexpression can be measured by any means known in the art. See, e.g.,Hollande et al., WO 2007/135542. Other genes whose expression orrepression is dependent on PG, such as ICAT, can also be assayed usingstandard methods. See id.

Pharmaceutical Compositions

Anti-PG monoclonal antibodies can be formulated in compositions.Optionally, the compositions can comprise one or more additionaltherapeutic agents, such as the second therapeutic agents describedbelow, are provided herein. The compositions will usually be supplied aspart of a sterile, pharmaceutical composition that will normally includea pharmaceutically acceptable carrier. This composition can be in anysuitable form (depending upon the desired method of administering it toa patient).

The anti-hPG monoclonal antibodies of the disclosure can be administeredto a patient by a variety of routes such as orally, transdermally,subcutaneously, intranasally, intravenously, intramuscularly,intraocularly, topically, intrathecally and intracerebroventricularly.The most suitable route for administration in any given case will dependon the particular antibody, the subject, and the nature and severity ofthe disease and the physical condition of the subject. The antibody canbe formulated as an aqueous solution and administered by subcutaneousinjection.

Pharmaceutical compositions can be conveniently presented in unit doseforms containing a predetermined amount of an anti-hPG monoclonalantibody of the disclosure per dose. Such a unit can contain for examplebut without limitation 5 mg to 5 g, for example 10 mg to 1 g, or 20 to50 mg. Pharmaceutically acceptable carriers for use in the disclosurecan take a wide variety of forms depending, e.g., on the condition to betreated or route of administration.

Pharmaceutical compositions of the disclosure can be prepared forstorage as lyophilized formulations or aqueous solutions by mixing theantibody having the desired degree of purity with optionalpharmaceutically-acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are referred to herein as“carriers”), i.e., buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants, and othermiscellaneous additives. See, Remington's Pharmaceutical Sciences, 16thedition (Osol, ed. 1980). Such additives must be nontoxic to therecipients at the dosages and concentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They can be present at concentration rangingfrom about 2 mM to about 50 mM. Suitable buffering agents for use withthe present disclosure include both organic and inorganic acids andsalts thereof such as citrate buffers (e.g., monosodium citrate-disodiumcitrate mixture, citric acid-trisodium citrate mixture, citricacid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additionally, phosphate buffers, histidinebuffers and trimethylamine salts such as Tris can be used.

Preservatives can be added to retard microbial growth, and can be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present disclosure include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalconium halides (e.g., chloride, bromide, and iodide),hexamethonium chloride, and alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.Isotonicifiers sometimes known as “stabilizers” can be added to ensureisotonicity of liquid compositions of the present disclosure and includepolyhydric sugar alcohols, for example trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol. Stabilizers refer to a broad category of excipients which canrange in function from a bulking agent to an additive which solubilizesthe therapeutic agent or helps to prevent denaturation or adherence tothe container wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thio sulfate; low molecular weight polypeptides (e.g.,peptides of 10 residues or fewer); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers,such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose,fructose, glucose; disaccharides such as lactose, maltose, sucrose andtrisaccacharides such as raffinose; and polysaccharides such as dextran.Stabilizers can be present in the range from 0.1 to 10,000 weights perpart of weight active protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) canbe added to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stressedwithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188,etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20,TWEEN®-80, etc.). Non-ionic surfactants can be present in a range ofabout 0.05 mg/ml to about 1.0 mg/ml, for example about 0.07 mg/ml toabout 0.2 mg/ml.

Additional miscellaneous excipients include bulking agents (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents.

Anti-PG monoclonal antibodies can be administered singly, as mixtures ofone or more anti-PG monoclonal antibodies, in mixture or combinationwith other agents useful for treating CRC, or adjunctive to othertherapy for CRC. Examples of suitable combination and adjunctivetherapies are provided below.

Encompassed by the present disclosure are pharmaceutical kits containingneutralizing anti-hPG monoclonal antibodies (including antibodyconjugates) of the disclosure. The pharmaceutical kit is a packagecomprising a neutralizing anti-hPG monoclonal antibody (e.g., either inlyophilized form or as an aqueous solution) and one or more of thefollowing:

-   -   A second therapeutic agent, for example as described below;    -   A device for administering the anti-hPG monoclonal antibody, for        example a pen, needle and/or syringe; and    -   Pharmaceutical grade water or buffer to resuspend the antibody        if the antibody is in lyophilized form.

Each unit dose of the anti-hPG monoclonal antibody can be packagedseparately, and a kit can contain one or more unit doses (e.g., two unitdoses, three unit doses, four unit doses, five unit doses, eight unitdoses, ten unit doses, or more). In a specific embodiment, the one ormore unit doses are each housed in a syringe or pen.

Effective Dosages

Neutralizing anti-PG monoclonal antibodies, or compositions thereof,will generally be used in an amount effective to achieve the intendedresult, for example an amount effective to treat CRC in a subject inneed thereof. Pharmaceutical compositions comprising neutralizinganti-PG monoclonal antibodies can be administered to patients (e.g.,human subjects) at therapeutically effective dosages. As used herein, a“therapeutically effective” dosage is an amount that confers atherapeutic benefit. In the context of CRC therapy, a therapeuticbenefit means any amelioration of CRC, including any one of, orcombination of, halting or slowing the progression of CRC (e.g., fromone stage of colorectal cancer to the next), halting or delayingaggravation or deterioration of the symptoms or signs of CRC, reducingthe severity of CRC, inducing remission of CRC, inhibiting CRC tumorcell proliferation, CRC tumor size, or CRC tumor number, or reducing PGserum levels.

The amount of neutralizing anti-PG monoclonal antibody administered willdepend on a variety of factors, including the nature and stage of theCRC being treated, the form, route and site of administration, thetherapeutic regimen (e.g., whether a second therapeutic agent is used),the age and condition of the particular subject being treated, thesensitivity of the patient being treated to anti-PG monoclonalantibodies. The appropriate dosage can be readily determined by a personskilled in the art. Ultimately, a physician will determine appropriatedosages to be used. This dosage can be repeated as often as appropriate.If side effects develop the amount and/or frequency of the dosage can bealtered or reduced, in accordance with normal clinical practice. Theproper dosage and treatment regimen can be established by monitoring theprogress of therapy using conventional techniques known to the peopleskilled of the art.

Effective dosages can be estimated initially from in vitro assays. Forexample, an initial dose for use in animals may be formulated to achievea circulating blood or serum concentration of anti-PG monoclonalantibody that is at or above the binding affinity of the antibody forprogastrin as measured in vitro. Calculating dosages to achieve suchcirculating blood or serum concentrations taking into account thebioavailability of the particular antibody is well within thecapabilities of skilled artisans. For guidance, the reader is referredto Fingl & Woodbury, “General Principles” in Goodman and Gilman's ThePharmaceutical Basis of Therapeutics, Chapter 1, latest edition,Pagamonon Press, and the references cited therein.

Initial dosages can be estimated from in vivo data, such as animalmodels. Animal models useful for testing the efficacy of compounds totreat CRC are well known in the art. Additionally, animal models of CRCare described in the Examples below. Ordinarily skilled artisans canroutinely adapt such information to determine dosages suitable for humanadministration.

The effective dose of a neutralizing anti-hPG monoclonal antibody of thedisclosure can range from about 0.001 to about 75 mg/kg per single(e.g., bolus) administration, multiple administrations or continuousadministration, or to achieve a serum concentration of 0.01-5000 μg/mlserum concentration per single (e.g., bolus) administration, multipleadministrations or continuous administration, or any effective range orvalue therein depending on the condition being treated, the route ofadministration and the age, weight and condition of the subject. In acertain embodiment, each dose can range from about 0.5 μg to about 50 μgper kilogram of body weight, for example from about 3 μg to about 30 μgper kilogram body weight.

Amount, frequency, and duration of administration will depend on avariety of factors, such as the patient's age, weight, and diseasecondition. A therapeutic regimen for administration can continue for 2weeks to indefinitely, for 2 weeks to 6 months, from 3 months to 5years, from 6 months to 1 or 2 years, from 8 months to 18 months, or thelike. Optionally, the therapeutic regimen provides for repeatedadministration, e.g., once daily, twice daily, every two days, threedays, five days, one week, two weeks, or one month. The repeatedadministration can be at the same dose or at a different dose. Theadministration can be repeated once, twice, three times, four times,five times, six times, seven times, eight times, nine times, ten times,or more. A therapeutically effective amount of anti-hPG monoclonalantibody can be administered as a single dose or over the course of atherapeutic regimen, e.g., over the course of a week, two weeks, threeweeks, one month, three months, six months, one year, or longer.

Therapeutic Methods

The ability of neutralizing anti-hPG monoclonal antibodies of thepresent disclosure to block PG-dependent responses, including cellproliferation, makes them useful for treating colorectal cancer.Accordingly, in another aspect, the present disclosure provides methodsof treating CRC in a patient in need thereof. Generally, the methodscomprise administering to the patient a therapeutically effective amountof a neutralizing anti-hPG monoclonal antibody of the disclosure.

A “subject” or “patient” to whom the anti-hPG monoclonal antibody of thedisclosure is administered is preferably a mammal such as a non-primate(e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkeyor human). The subject or patient can be a human, such as an adultpatient or a pediatric patient.

Patients suitable for anti-hPG monoclonal antibody therapy are patientsdiagnosed with CRC. The CRC can be of any type and at any clinical stageor manifestation. Suitable subjects include patients with CRC tumors(operable or inoperable), patients whose tumors have been surgicallyremoved or resected, patients with a CRC tumor comprising cells carryinga mutation in an oncogene, such as, for example, RAS or APC, patientswho have received or receive other therapy for CRC in combination withor adjunctive to anti-hPG monoclonal antibody therapy. Other therapy forCRC includes, but is not limited to, chemotherapeutic treatment,radiation therapy, surgical resection, and treatment with one or moreother therapeutic antibodies, as detailed below.

Anti-hPG monoclonal antibody therapy can be combined with, or adjunctiveto, one or more other treatments. Other treatments include, withoutlimitation, chemotherapeutic treatment, radiation therapy, surgicalresection, and antibody therapy, as described herein.

Anti-hPG monoclonal antibody therapy can be adjunctive to othertreatment, including surgical resection.

Combination therapy as provided herein involves the administration of atleast two agents to a patient, the first of which is a neutralizinganti-hPG monoclonal antibody of the disclosure, and the second of whichis a second therapeutic agent. The neutralizing anti-hPG monoclonalantibody and the second therapeutic agent can be administeredsimultaneously, successively, or separately.

As used herein, the neutralizing anti-hPG monoclonal antibody and thesecond therapeutic agent are said to be administered successively ifthey are administered to the patient on the same day, for example duringthe same patient visit. Successive administration can occur 1, 2, 3, 4,5, 6, 7 or 8 hours apart. In contrast, the anti-hPG monoclonal antibodyof the disclosure and the second therapeutic agent are said to beadministered separately if they are administered to the patient on thedifferent days, for example, the anti-hPG monoclonal antibody of thedisclosure and the second therapeutic agent can be administered at a1-day, 2-day or 3-day, one-week, 2-week or monthly intervals. In themethods of the present disclosure, administration of the anti-hPGmonoclonal antibody of the disclosure can precede or followadministration of the second therapeutic agent.

As a non-limiting example, the neutralizing anti-hPG monoclonal antibodyand second therapeutic agent can be administered concurrently for aperiod of time, followed by a second period of time in which theadministration of the anti-hPG monoclonal antibody of the disclosure andthe second therapeutic agent is alternated.

Combination therapies of the present disclosure can result in a greaterthan additive, or a synergistic, effect, providing therapeutic benefitswhere neither the neutralizing anti-hPG monoclonal antibody nor secondtherapeutic agent is administered in an amount that is, alone,therapeutically effective. Thus, such agents can be administered inlower amounts, reducing the possibility and/or severity of adverseeffects.

A second therapeutic agent can be a chemotherapeutic agent.Chemotherapeutic agents include, but are not limited to, radioactivemolecules, toxins, also referred to as cytotoxins or cytotoxic agents,which includes any agent that is detrimental to the viability of cells,agents, and liposomes or other vesicles containing chemotherapeuticcompounds. Examples of suitable chemotherapeutic agents include but arenot limited to 1-dehydrotestosterone, 5-fluorouracil decarbazine,6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin, aldesleukin,alkylating agents, allopurinol sodium, altretamine, amifostine,anastrozole, anthramycin (AMC)), anti-mitotic agents,cis-dichlorodiamine platinum (II) (DDP) cisplatin), diamino dichloroplatinum, anthracyclines, antibiotics, antimetabolites, asparaginase,BCG live (intravesical), betamethasone sodium phosphate andbetamethasone acetate, bicalutamide, bleomycin sulfate, busulfan,calcium leucouorin, calicheamicin, capecitabine, carboplatin, lomustine(CCNU), carmustine (BSNU), Chlorambucil, Cisplatin, Cladribine,Colchicin, conjugated estrogens, Cyclophosphamide, Cyclothosphamide,Cytarabine, Cytarabine, cytochalasin B, Cytoxan, Dacarbazine,Dactinomycin, dactinomycin (formerly actinomycin), daunirubicin HCL,daunorucbicin citrate, denileukin diftitox, Dexrazoxane,Dibromomannitol, dihydroxy anthracin dione, Docetaxel, dolasetronmesylate, doxorubicin HCL, dronabinol, E. coli L-asparaginase, emetine,epoetin-α, Erwinia L-asparaginase, esterified estrogens, estradiol,estramustine phosphate sodium, ethidium bromide, ethinyl estradiol,etidronate, etoposide citrororum factor, etoposide phosphate,filgrastim, floxuridine, fluconazole, fludarabine phosphate,fluorouracil, flutamide, folinic acid, gemcitabine HCL, glucocorticoids,goserelin acetate, gramicidin D, granisetron HCL, hydroxyurea,idarubicin HCL, ifosfamide, interferon α-2b, irinotecan HCL, letrozole,leucovorin calcium, leuprolide acetate, levamisole HCL, lidocaine,lomustine, maytansinoid, mechlorethamine HCL, medroxyprogesteroneacetate, megestrol acetate, melphalan HCL, mercaptipurine, mesna,methotrexate, methyltestosterone, mithramycin, mitomycin C, mitotane,mitoxantrone, nilutamide, octreotide acetate, ondansetron HCL,oxaliplatin, paclitaxel, pamidronate disodium, pentostatin, pilocarpineHCL, plimycin, polifeprosan 20 with carmustine implant, porfimer sodium,procaine, procarbazine HCL, propranolol, rituximab, sargramostim,streptozotocin, tamoxifen, taxol, tegafur, teniposide, tenoposide,testolactone, tetracaine, thioepa chlorambucil, thioguanine, thiotepa,topotecan HCL, toremifene citrate, trastuzumab, tretinoin, valrubicin,vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate.

The neutralizing anti-hPG monoclonal antibodies disclosed herein can beadministered to a patient in need of treatment for colorectal cancerreceiving a combination of chemotherapeutic agents. Exemplarycombinations of chemotherapeutic agents include 5-fluorouracil (5FU) incombination with leucovorin (folinic acid or LV); capecitabine, incombination with uracil (UFT) and leucovorin; tegafur in combinationwith uracil (UFT) and leucovorin; oxaliplatin in combination with 5FU,or in combination with capecitabine; irinotecan in combination withcapecitabine, mitomycin C in combination with 5FU, irinotecan orcapecitabine. Use of other combinations of chemotherapeutic agentsdisclosed herein is also possible.

As is known in the relevant art, chemotherapy regimes for colorectalcancer using combinations of different chemotherapeutic agents have beenstandardized in clinical trials. Such regimes are often known byacronyms and include 5FU Mayo, 5FU Roswell Park, LVFU2, FOLFOX, FOLFOX4,FOLFOX6, bFOL, FUFOX, FOLFIRI, IFL, XELOX, CAPDX, XELIRI, CAPIRI,FOLFOXIRI. See, e.g., Chau, I., et al., 2009, Br. J. Cancer 100:1704-19and Field, K., et al., 2007, World J. Gastroenterol. 13:3806-15, both ofwhich are incorporated by reference.

Neutralizing anti-hPG monoclonal antibodies can also be combined withother therapeutic antibodies. Accordingly, anti-hPG monoclonal antibodytherapy can be combined with, or administered adjunctive to a differentmonoclonal antibody such as, for example, but not by way of limitation,an anti-EGFR (EGF receptor) monoclonal antibody or an anti-VEGFmonoclonal antibody. Specific examples of anti-EGFR antibodies includecetuximab and panitumumab. A specific example of an anti-VEGF antibodyis bevacizumab.

Detection of Progastrin Using Anti-hPG Antibodies

Anti-PG monoclonal antibodies, whether neutralizing or non-neutralizing,are also useful for applications that depend on PG detection such asdiagnosing CRC or monitoring the effects of treatment on a subject'sCRC. Accordingly, in an aspect, the present disclosure provides a methodof diagnosing colorectal cancer in a patient, comprising determining theamount of progastrin in a sample from the patient using an anti-hPGmonoclonal antibody according to the present disclosure. Generally,methods of diagnosing colorectal cancer in a patient comprise measuringprogastrin in a sample obtained from a patient using the anti-hPGmonoclonal antibodies of the disclosure, wherein a measurement of 20 pMto 400 pM of progastrin in the sample is indicative of colorectalcancer. Progastrin can be measured in samples of, e.g., blood, serum,plasma, tissue, and/or cells. hPG detection can be carried out usingassays known in the art and/or described herein, such as, ELISA,sandwich ELISA, immunoblotting (Western blotting), immunoprecipitation,BIACORE technology and the like.

As noted herein, progastrin is but one of a number of differentpolypeptides resulting from post-translational processing of the gastringene product. Diagnostic, monitoring and other methods described hereinspecifically detect hPG as opposed to other gastrin gene products,including degradation products. Accordingly, in specific embodiments,hPG is detected using an ELISA as disclosed herein, wherein twoantibodies to hPG are used, targeting the N- and C-terminus of hPGrespectively. In some embodiments, one of the two antibodies used fordetection is an anti-hPG monoclonal antibody as described herein. hPGlevels ranging from 20 pM to 400 pM are indicative of colorectal cancer.

In general, the procedure for determining hPG levels using anti-hPGmonoclonal antibodies is as follows. A surface, such as the wells in a96-well plate, is prepared to which a known quantity of a first,“capture,” antibody to hPG is bound. The capture antibody can be, forexample, an anti-hPG antibody which binds with to a C- or N-terminalregion of hPG. After blocking, a test sample is applied to the surfacefollowed by an incubation period. The surface is then washed to removeunbound antigen and a solution containing a second, “detection,”antibody to hPG is applied. The detection antibody can be any of theanti-hPG monoclonal antibodies described herein, provided the detectionantibody binds a different epitope from the capture antibody. Forexample, if the capture antibody binds a C-terminal peptide region ofhPG, then a suitable detection antibody would be one that binds anN-terminal peptide region of hPG. Progastrin levels can then be detectedeither directly (if, for example, the detection antibody is conjugatedto a detectable label) or indirectly (through a labeled secondaryantibody that binds the detection anti-hPG antibody).

In a specific embodiment, hPG levels are measured as follows from a testsample. 96-well microtiter plates are coated with between 0.5 and 10μg/mL of a rabbit C-terminal anti-hPG polyclonal antibody and incubatedovernight. Plates are then washed three times in PBS-Tween (0.05%) andblocked with 2% (w/v) nonfat dried milk in PBS-Tween (0.05%).Separately, test samples, control samples (blank or PG-negative plasmaor serum samples), and between about 5 μM (0.5×10⁻¹¹M) and about 0.1 nM(1×10⁻¹⁰ M) of an hPG reference standard (lyophilized hPG diluted inPG-negative plasma or serum) are prepared in an appropriate diluent(e.g., PBS-Tween 0.05%). Samples are incubated on the coated plates forbetween 2 and 4 hours at 37° C., or alternatively between 12 and 16hours at 21° C. After incubation, plates are washed three times withPBS-Tween (0.05%) and incubated with between 0.001 and 0.1 μg/mL of anN-terminal anti-hPG monoclonal antibody as described herein, coupled tohorseradish peroxidase (HRP) (Nakane et al., 1974, J. Histochem.Cytochem. 22(12): 1084-1091) for 30 minutes at 21° C. Plates are thenwashed three times in PBS-Tween (0.05%) and HRP substrate is added for15 minutes at 21° C. The reaction is stopped by added 100 μL of 0.5Msulfuric acid and an optical density measurement taken at 405 nm. Testsample hPG levels are determined by comparison to a standard curveconstructed from the measurements derived from the hPG referencestandard.

Typically, patients are diagnosed based on invasive procedures such ashistological assessment of biopsied tissue as well as other invasiveprocedures such as colonoscopy. CRC is divided into 5 stages rangingfrom Stage 0 (cancer limited to innermost lining of colon or rectum),Stage 1 (cancer in inner wall of colon or rectum), Stage 2 (cancerextended through wall of colon but not found in adjacent lymph nodes),Stage 3 (cancer found in lymph nodes and tissue surrounding colon orrectum), and Stage 4 (cancer has spread to other parts of the body).From a histological perspective, colorectal tumors present with a broadspectrum of neoplasms, ranging from benign growths to invasive cancer,and are predominantly epithelial-derived tumors (i.e., adenomas oradenocarcinomas). Lesions can be classified into three groups:normeoplastic polyps, neoplastic polyps (adenomatous polyps, adenomas),and cancers. Adenomatous polyps are benign tumors that may undergomalignant transformation, and have been classified into three histologictypes, with increasing malignant potential: tubular, tubulovillous, andvillous. Adenocarcinomas have also been categorized according to theirhistology into mucinous (colloid) adenocarcinoma; signet ringadenocarcinoma; scirrhous tumors; and neuroendocrine.

In contrast to current means for diagnosing CRC, the present disclosureprovides methods for diagnosing subjects with CRC in the absence of anyhistological analysis or disease staging, based on measurement of hPGlevels that can be determined from a blood sample. Furthermore, methodsof the present disclosure are useful in selecting CRC patients suited toanti-hPG monoclonal therapy regardless of how a patient has beendiagnosed.

Serum PG levels are also useful in assessing efficacy of CRC treatment.Accordingly, the present disclosure provides a method for monitoring theeffectiveness of colorectal cancer therapy comprising determining PGlevels in a patient being treated for CRC. Methods for monitoring theeffectiveness of colorectal cancer therapy comprise repeatedlydetermining hPG levels using an anti-PG monoclonal antibody of thepresent disclosure in a colorectal cancer patient undergoing treatmentfor colorectal cancer, wherein a decrease in the patient's circulatinghPG levels over an interval of treatment is indicative of treatmentefficacy. For example, a first measurement of a patient's circulatinghPG levels can be taken followed by a second measurement while or afterthe patient receives treatment for colorectal cancer. The twomeasurements are then compared, and a decrease in hPG levels isindicative of therapeutic benefit.

In an aspect, the disclosure provides diagnostic kits containing theanti-hPG monoclonal antibodies (including antibody conjugates). Thediagnostic kit is a package comprising at least one anti-hPG monoclonalantibody of the disclosure (e.g., either in lyophilized form or as anaqueous solution) and one or more reagents useful for performing adiagnostic assay (e.g., diluents, a labeled antibody that binds to ananti-hPG monoclonal antibody, an appropriate substrate for the labeledantibody, hPG in a form appropriate for use as a positive control andreference standard, a negative control). In specific embodiments, a kitcomprises two anti-hPG antibodies, wherein at least one of theantibodies is an anti-hPG monoclonal antibody. Optionally, the secondantibody is a polyclonal anti-hPG antibody. In some embodiments, the kitof the present disclosure comprises an N-terminal anti-hPG monoclonalantibody as described herein.

Anti-hPG antibodies can be labeled, as described above. Alternatively,the kit can include a labeled antibody which binds an anti-hPGmonoclonal antibody and is conjugated to an enzyme. Where the anti-hPGmonoclonal antibody or other antibody is conjugated to an enzyme fordetection, the kit can include substrates and cofactors required by theenzyme (e.g., a substrate precursor which provides the detectablechromophore or fluorophore). In addition, other additives can beincluded, such as stabilizers, buffers (e.g., a block buffer or lysisbuffer), and the like. Anti-hPG monoclonal antibodies included in adiagnostic kit can be immobilized on a solid surface, or, alternatively,a solid surface (e.g., a slide) on which the antibody can be immobilizedis included in the kit. The relative amounts of the various reagents canbe varied widely to provide for concentrations in solution of thereagents which substantially optimize the sensitivity of the assay.Antibodies and other reagents can be provided (individually or combined)as dry powders, usually lyophilized, including excipients which ondissolution will provide a reagent solution having the appropriateconcentration.

Kits may include instructional materials containing instructions (e.g.,protocols) for the practice of diagnostic methods. While theinstructional materials typically comprise written or printed materials,they are not limited to such. A medium capable of storing suchinstructions and communicating them to an end user is contemplated bythis invention. Such media include, but are not limited to, electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

10. EXAMPLES

The following examples are illustrative and not intended to be limiting.

Example 1 Generation of Monoclonal Antibodies Against ProgastrinImmunogens for Human Progastrin

Several immunogens were generated to develop hybridomas producingmonoclonal antibodies against human progastrin. Antigens previously usedto generate polyclonal antibodies, such as full length human progastrinand immunogens based on residues 70 to 80 of hPG, failed to lead to amonoclonal immune response or give rise to PG-specific monoclonalantibodies. As described in more detail below, antigens 14 amino acidsand longer, which included sequences unique to hPG at either theN-terminal and C-terminal end of the protein, were capable of inducingan adequate immune response in animals and were used to generatehybridomas producing over 20 different monoclonal antibodies to hPG.Surprisingly, several immunogens that included residues 55 to 80 of hPG,some of which are also found in other gastrin gene-derived peptides, wasused successfully to generate monoclonal antibodies that were specificto hPG. The table below summarizes the immunogens tested.

TABLE 2 No. of Experi- positive ment Immunogen clones 1Human progastrin (SEQ ID NO: 20)  0* 1 (SEQ ID NO: 26)-Ahx-Cys-BSA 2 2(SEQ ID NO: 26)-Ahx-Cys-KLH 2 3 (SEQ ID NO: 26)-Ahx-Cys-KLH 8 1BSA-Cys-Ahx-Ahx-(SEQ ID NO: 97) 0 2 KLH-Cys-Ahx-Ahx-(SEQ ID NO: 97) 0 3KLH-Cys-Ahx-Ahx-(SEQ ID NO: 96) 10 DT-Cys-Ahx-Ahx-(SEQ ID NO: 96) 3 *Theimmunized mice did not display any immune response.

The immunogens listed in Table 2 were made according to techniques knownin the art, using chemical synthesis of the peptide sequence and thelinker, followed by crosslinking to the Bovine Serum Albumin (BSA),Keyhole Limpet Hemocyanin (KLH), or Diptheria Toxin (DT) carrier usingan appropriate crosslinking agent (e.g., MBS(m-Maleimidobenzoyl-N-hydrosuccinimide ester), glutaraldehyde orSulfo-SMCC (Sulfosuccinimidyl4-[N-maleimidomethyl]cyclolhexane-1-carboxylate). (Coligan J E et al.,Current protocols in Immunology, Vol. 2, New York: John Wiley and Sons;1996, p 9.0.1-9.0.15; Harlow D L. Antibodies: A Laboratory Manual. NewYork: Cold Spring Harbor Laboratory; 1998, p 72-87). Linkers used wereone or two aminohexanoic acid (Ahx) residues coupled to a cysteineresidue.

In each of three experiments, Balb/c mice were injected 4 to 5 times forN-terminal immunogens, and 2 to 4 times for C-terminal immunogens. Eachinjection administered 10 μg of the immunogen with Ribi, Alun orFreund's adjuvant.

Cell Fusions and Hybridoma Screening

Serum from each mouse was tested by ELISA against the immunogen andsplenocytes harvested from the mice with the strongest immune response.Splenocytes were fused with Sp2 myeloma cells using polyethylene glycol,and seeded into 96-well plates at a density of 15,000 to 35,000 cellsper well. Fused cells were selected for, using medium containinghypoxanthine, aminopterin, and thymidine (HAT medium).

Supernatants of hybridomas were screened by ELISA for the ability tobind the immunogen and full length progastrin. Three rounds of screeningwere performed to ensure that only hybridomas stably producingantibodies recognizing full length hPG and the immunogen were selected.

Screening of hybridomas and monoclonal antibodies to determine bindingto different PG peptides was performed using an ELISA technique asdescribed below. This protocol was used to screen for PG binding offused splenocytes, first and second round sub-clones, as well as forverifying the specificity of the antibodies to PG, as compared to othergastrin gene-derived peptides.

Briefly, 96-well plates were incubated overnight at 4° C. withappropriate concentration(s) of a test peptide in Phosphate-BufferedSaline (PBS), after which the wells were washed three times with washsolution (PBS and 0.1% Tween-20), and incubated for 2 hours at 22° C.with 100 μl, blocking solution (PBS, 0.1% Tween-20, 0.1% Bovine SerumAlbumin or casein hydrolysate) per well. After blocking, the wells werewashed three times and the primary antibody—the antibody to beassayed—added. For initial screening of fused splenocytes, 50 μL ofculture supernatant from each culture to be assayed was added to eachwell in the plate. In assays performed on monoclonal antibodies, 100 μLof the test antibody in PBS and 0.1% Tween-20 was added to each well.Plates were then incubated for 2 hours at 22° C., after which theprimary antibody solution was discarded and replaced, after a wash step(3×100 μL wash solution, as noted above), with blocking solutioncontaining the secondary antibody, which binds the primary antibody andis coupled to an enzyme. The secondary antibody was a goat anti-mouseIgG (Fc) antibody coupled to horseradish peroxidase. After a 1-hourincubation with secondary antibody, 100 μL of substrate solution (e.g.Fast OPD, or O-Phenylenediamine dihydrochloride, available fromSigma-Aldrich Co., prepared according to manufacturer's directions) wasadded to each well and incubated in the dark for 20 minutes at 22° C.The reaction was then stopped by adding 50 μL of 4N sulfuric acid andthe amount of substrate catalyzed determined by measuring the opticaldensity (O.D.) at 492 nm. Substrate conversion is proportional to theamount of primary (test) antibody bound to the antigen. Experiments wererun in duplicate and OD measurements plotted as a function of antigen orantibody concentration depending on the goal of the experiment. Sampleswere scored as positive for anti-PG antibodies if the measured O.D. wasbetween 0.2 and 1.5. The same O.D. bracket was used to identifyantibodies that bound the immunogenic peptide used to inoculate testanimals.

Exemplary materials and reagents used in the assay are as follows:

Item Source Reference Greiner Microlon 96-well plates Dutscher # 65509210X DPBS Dutscher # P04-53500 Tween-20 Sigma # 63158 BSA (for blocking)Euromedex # 04-100-810-C Hydrolyzed casein (when used instead Sigma22090 of BSA) Hybridoma supernatants or N-terminal BioRéalités Asdescribed or C-terminal monoclonal antibodies herein Goat anti-mouse IgG(Fc), Thermo # 31439 peroxidase-coupled Fast OPD Sigma # P9187 95-97%Sulfuric Acid Sigma # 30743

Exemplary peptides used in screening of hybridomas and monoclonalantibodies were as follows:

Screening peptides Source Reference BSA Euromedex # 04-100-810-0Recombinant human progastrin BioRéalités McQueen et al., 2002, J.Protein Chem., 21(7): 465-471. Human gastrin I (G-17) Sigma # 53673Glycine-extended gastrin Auspep # 810082 17 (G-Gly) KLH Pierce (Perbio)# 77653 C-terminal flanking Auspep # R41345 peptide (CTFP)Recombinant human progastrin was produced as described in McQueen et al,2002, J. Protein Chem. 21: 465-471) with minor modifications. Briefly,BL21 DE3 Star bacterial cells (InVitrogen) were transformed with avector containing the full-length human progastrin sequence in aPGEX-GST-TEV backbone (GE Healthcare). Bacteria were grown in LB mediumcontaining 0.5 mM IPTG for 3 hours at 37° C. Bacterial pellets werebroken using a French Press, and soluble as well as non-solublefractions were separated by centrifugation. Thereafter, GST-tagged hPGwas isolated using a glutathione affinity column and PG was cleaved fromGST with the Tobacco Etch Virus NIa (TEV) protease. Finally, PG wasdialyzed against the final buffer (10 mM Hepes, 0.5% BSA, pH 7.4).

Hybridomas were first cloned, then subcloned and amplified. Positivehybridomas were selected based on the following criteria: (1) PG andimmunogen specificity, (2) relative affinity of antibodies, (3)hybridoma cell growth, (4) antibody secretion, and (5) monoclonality ofhybridomas. Assays and selection criteria were as follows.

For specificity, supernatants of test hybridomas were assayed by ELISAas described above (50 μL of a 1 in 2 dilution of supernatant into assaymedium PBS). Hybridomas were scored as positive if the O.D. measurementwas between 0.2 and 1.5 in an assay with hPG or the immunogen used toinoculate test mice. As a further criterion for specificity, clones wereselected based on a lack of binding to other gastrin gene-derivedpeptides. Lack of binding was measured as no statistically significantlydifference between the signal from test wells and the average signalfrom control wells containing only PBS.

For affinity characterization, serial dilutions of antibodies wereassayed by ELISA for binding to hPG, as described above. Standarddilutions used in assays of N-terminal antibodies were 0, 0.1, 0.3, 1,3, 10, 30, 100 ng/ml. Standard dilutions used in assays of C-terminalantibodies were 0, 0.03, 0.1, 0.3, 1, 3, 10, 30 ng/ml.

Hybridoma cell growth through multiple rounds of serial culture wasassessed by microscopic observation two days after seeding. Cells areexpected to proliferate and fill the well by 48 hours after seeding.Serial dilution (typically, a first dilution at 1:5, followed by atleast 2 further dilutions at 1:10) was performed, followed bymicroscopic observation at 48 hours to confirm adequate growth. Cellsfrom hybridomas that fulfill this criterion were diluted and seededagain, to be observed under the microscope 48 h later. Such rounds of“dilution-seeding-observation” were repeated 3 times before a hybridomawas scored as fulfilling the “growth” criteria.

Antibody secretion was tested by performing ELISAs using hPG asdescribed above on serial dilutions (1/2, 1/20, 1/200, 1/500, 1/1000,1/2000) of cell-free supernatants.

Monoclonality was determined by seeding a clone or hybridoma in a96-well plate at a density of 0.6 cells/well and incubating for twoweeks. At two weeks, supernatants were assayed for hPG binding by ELISAand the clonal nature of the population determined based on having aconsistent OD value across at least 90% of live-cell containing wells.

Monoclonal Antibodies Against Full Length Progastrin

Each of three mice was inoculated with recombinant human progastrin(described above). The immunogen failed to elicit any detectable immuneresponse in the mice: no binding to PG could be detected using an ELISAas described above. No fusions were performed.

N-Terminal Monoclonal Antibodies Against Progastrin

As noted above, N-terminal monoclonal antibodies were generated againstan antigen containing residues 1 to 14 of hPG linked to either bovineserum albumin (BSA) or keyhole limpet hemocyanin (KLH) by way of anAhx-Cys linker on the C-terminal end of the 14 residue antigen(SWKPRSQQPDAPLG-Ahx-Cys (SEQ ID NO:26)).

In a first experiment, three mice were inoculated with a BSA-linkedN-terminal peptide. From three fusions, performed with splenocytes fromtwo of the three mice, one fusion yielded no clones that showed bindingto PG or the immunogen. Of the two other fusions seeded in 96-wellplates, one generated 4 PG-binding and PG-specific hybridomas, fromwhich a single stable, IgG producing subclone was recovered. The secondfusion also resulted in a single stable IgG1-producing, PG-binding andPG-specific hybridoma subclone. Overall, from three mice, 17 first roundhybridomas, or 0.74% of the hybridomas screened) were positive for PG-and immunogen-binding, from which 9 positive cell lines were subcloned,of which two were positive, IgG-producing cell lines. Thus, the firstexperiment generated two clones after a couple of rounds of subcloningthat retained a strong positive signal against the immunogen and hPG(positive for “PG-binding”) and did not bind other gastrin gene-derivedpeptides (positive for “PG-specific”): hybridomas 43B9G11 and WE5H2G7,producing anti-hPG MAb1 and anti-hPG MAb 2 respectively.

In a second experiment, mice were inoculated with a KLH-linkedN-terminal peptide. Two fusions were performed with Sp2 myeloma cells.Of these, only one fusion generated PG- and immunogen-positive clones,that were also PG-specific. From this, 1920 hybridomas were seeded. Manyhybridomas tested positive with the immunizing peptides but notprogastrin, or were not PG-specific. Specifically, 297 hybridomas showeda strong positive signal for the immunizing peptide (around 15.5% of1920), of which 124 were also positive for progastrin binding (6.5%).There were 36 hybridomas, or 1.8%, that were positive for progastrin butnot the immunogen used. Only 12 hybridomas of the 1920 seeded yieldedantibodies that specifically bind progastrin but not other gastrin geneproducts (0.6% of total hybridomas, 3.6% of clones that were positivefor peptide and/or progastrin on first screening). Of the 12 selectedclones, only 2 were stable enough to be established as permanent clonesand frozen for long-term storage. Thus, in this second experiment, ofthe almost 2000 hybridomas were seeded, only 2 clones, 6B5B11C10(producing anti-hPG MAb 3) and 20D2C3G2 (producing anti-hPG MAb4),producing anti-hPG MAb3 and MAb4 respectively, were recovered thatexpress monoclonal antibodies capable of binding hPG and the immunizingpeptide, and having specificity to progastrin over other gastrin geneproducts and high affinity to hPG. Both exemplary antibodies are of theIgG1 isotype.

In a third experiment, mice were inoculated with the same immunogen asin the second experiment. Fusions to Sp2 myeloma cells were performedwith splenocytes from the two mice with the strongest immune response.Hybridomas were seeded from the fusions (3840 hybridomas from one fusioneach, per mouse) and supernatants tested for PG- and immunogen-binding,as well as PG-specificity. From each fusion, 6 hybridomas showedPG-specificity, from which 3 subclones were selected that met thefurther selection for growth, monoclonality, antibody secretion,relative affinity. Thus, in all, 2.9% of the hybridomas tested afterseeding (220/7680) were PG and immunogen-positive, of which 0.15% werepositive clones (12/7680), the final subclones recovered constituting0.15% of the original hybridomas seeded (6/7680). This experimentgenerated hybridomas 1E9A4A4 (producing anti-hPG MAb 15), 1E9D9B6(producing anti-hPG MAb 16), 1C8D10F5 (producing anti-hPG MAb 17),1A7C3F11 (producing anti-hPG MAb 18), 1B3B4F11 (producing anti-hPG MAb19), and 1C11F5E8 (producing anti-hPG MAb 20).

The table below shows the number of clones seeded for each experiment,the immunogen used, and the number and percentage of hybridomasproducing monoclonal antibodies that recognized both the immunogen andfull length progastrin.

TABLE 3 PG specific, PG⁺ hybridoma PG specific IgG producing ExptImmunogen Clones seeded supernatants clones subclones 1(SEQ ID NO: 26)-Ahx- 2304  17 (0.74%)  9 (0.39%) 2 (0.087%) Cys-BSA 2(SEQ ID NO: 26)-Ahx- 1920 124 (6.5%) 12 (0.6%) 2 (0.1%) Cys-KLH 3(SEQ ID NO: 26)-Ahx- 7680 220 (2.9%) 12 (0.15%) 6 (0.1%) Cys-KLH

C-Terminal Monoclonal Antibodies Against Progastrin

C-terminal monoclonal antibodies were generated against an antigencontaining residues 55 to 80 of hPG linked to either KLH or DT by way ofan Cys-Ahx-Ahx linker on the N-terminal end of the 26 residue antigen(Cys-Ahx-Ahx-QGPWLEEEEEAYGWMDFGRRSAEDEN (SEQ ID NO:96)). Attempts togenerate hybridomas with a smaller antigen containing only residues 70to 80 of hPG, Cys-Ahx-Ahx-FGRRSAEDEN (SEQ ID NO:97), conjugated eitherto BSA or KLH, failed to generate any clones.

Three experiments were performed. In the first two experiments, in whichthe shorter peptide was used, zero subclones were recovered.Specifically, in a first experiment 4 mice were injected with aC-terminal peptide corresponding to SEQ ID NO:97, linked to BSA. None ofthe fusions generated any IgG-producing hybridomas. In a secondexperiment, 6 mice were injected, 3 each, with a peptide correspondingto SEQ ID NO:97, linked to KLH at its N-terminal end, or a peptidecorresponding to SEQ ID NO:97, linked to KLH at its C-terminal end. Forthe first immunogen, fusions were performed and hybridomas recovered,but no subclones positive for PG-binding and PG-specificity wereisolated. For the second immunogen, none of the mice developed an immuneresponse and no fusions were performed.

In a third experiment, a 26 amino acid peptide including C-terminalsequences not unique to hPG was used. The immunogen, which included apeptide corresponding to SEQ ID NO:96 linked to either KLH or DT, wasinjected into mice. Fusions to Sp2 myeloma cells were performed withsplenocytes from the two mice that had the strongest response. 3840hybridomas were seeded from one fusion per mouse. Overall, from 7680hybridomas screened, of which 382 (5%) were PG-positive and PG-specific,13 (0.17%) stable, positive subclones were recovered: 1B4A11D11(producing anti-hPG MAb 5), 1B6A11F2 (producing anti-hPG MAb 6),1B11E4B11 (producing anti-hPG MAb 7), 1C10D3B9 (producing anti-hPG MAb8), D8F5B3 (producing anti-hPG MAb 9), 1E1C7B4 (producing anti-hPG MAb10), 2B4C8C8 (producing anti-hPG MAb 11), 2B11E6G4 (producing anti-hPGMAb 12), 2C6C3C7 (producing anti-hPG MAb 13), 2H9F4B7 (producinganti-hPG MAb 14), 1F11F5E10 (producing anti-hPG MAb 21), 1F11F5G9(producing anti-hPG MAb 22), and 1A11F2C9 (producing anti-hPG MAb 23).

The table below shows the number of clones seeded for each experiment,the immunogen used, the number of hybridomas screened, the number andpercentage of hybridomas producing PG- and immunogen-binding monoclonalantibodies, that are PG-specific and meeting the selection criteria(growth, monoclonality, relative affinity, etc.) after three rounds ofsubcloning.

TABLE 4 PG⁻ specific, Clones PG⁺ hybridoma PG Specific IgG-producingExpt Immunogen seeded supernatants clones subclones 1 BSA-linker-(SEQ IDNO: 97) 3072 10 (0.32%)  9 (0.29%) 0 2 KLH- linker-(SEQ ID NO: 97) 192027 (0.47%)  4 (0.07%) 0 (SEQ ID NO: 97)- linker-KLH 0 0 0 3KLH-linker-(SEQ ID NO: 96) 3840 192 (5%)    17 (0.44%) 10 (0.26%)DT-linker-(SEQ ID NO: 96) 3840 190 (4.95%)  13 (0.34%)  3 (0.08%)

The monoclonal antibodies, and the stable hybridomas from which they areproduced are detailed in the table below:

TABLE 5 Monoclonal antibody Hybridoma Mouse Ig MAb 1 43B9G11 IgG1 MAb 2WE5H2G7 IgG1 MAb 3 6B5B11C10 IgG1 MAb 4 20D2C3G2 IgG1 MAb 5 1B4A11D11IgG1 MAb 6 1B6A11F2 IgG1 MAb 7 1B11E4B11 IgG1 MAb 8 1C10D3B9 IgG1 MAb 91D8F5B3 IgG1 MAb 10 1E1C7B4 IgG1 MAb 11 2B4C8C8 IgG1 MAb 12 2B11E6G4IgG1 MAb 13 2C6C3C7 IgG1 MAb 14 2H9F4B7 IgG1 MAb 15 1E9A4A4 IgG1 MAb 161E9D9B6 IgG1 MAb 17 1C8D10F5 N.D. MAb 18 1A7C3F11 IgG2 MAb 19 1B3B4F11IgG2 MAb 20 1C11F5E8 IgG2 MAb 21 1F11F5E10 IgG2 MAb 22 1F11F5G9 IgG2 MAb23 1A11F2C9 IgG2

Hybridoma cell lines 1B4A11D11 (MAb 5, registration no. CNCM I-4371),1B6A11F2 (MAb 6, registration no. CNCM I-4372), 1B11E4B11 (MAb 7,registration no. CNCM I-4373), 2B4C8C8 (MAb 11, registration no. CNCMI-4374), 2B11E6G4 (MAb 12, registration no. CNCM I-4375), and 1E9A4A4(MAb 15, registration no. and CNCM I-4376) were deposited in accordancewith the Treaty of Budapest and received on Oct. 6, 2010 by theCollection Nationale de Cultures de Microorganisms (CNCM), InstitutPasteur, Paris, France.

Cloning and Sequencing of Anti-hPG Monoclonal Antibodies

Monoclonal antibodies produced by hybridomas can be cloned and sequencedusing techniques known to those skilled in the art. Monoclonalantibodies from hybridomas listed in Table 5 above were sequenced asdescribed below.

Sequences encoding monoclonal antibodies produced by hybridomas6B5B11C10 and 20D2C3G2 were cloned and sequenced using standardtechniques. Briefly, total RNA was isolated from frozen cell pelletsusing RNABee reagent, AMSBio catalogue no. CS-104B, used according tomanufacturer's instructions. cDNA for V-regions was prepared from mRNAusing reverse-transcriptase polymerase chain reaction (RT-PCR), followedby 5′ rapid amplification of cDNA ends (RACE). cDNA synthesis wascarried out using constant-region-specific primers, after which thefirst strand product was purified and terminal deoxynucleotidetransferase was used to add homopolymeric tails to the 3′ ends of thecDNA. The “tailed” cDNA sequences were then amplified by PCR usingprimer pairs, one primer each for the homopolymeric tail and either theV_(H) or V_(L) region, respectively. Heavy and light chain variableregion PCR products were then cloned into a “TA” cloning vector (p-GEM-Teasy, Promega cat. no A 1360) and sequenced using standard procedures.See FIG. 2A-B (MAb 3), FIG. 2C-D (MAb 4).

Sequences encoding monoclonal antibodies produced by hybridomas1C10D3B9, 2C6C3C7, 1B3B4F1, and 1E9D9B61 were determined as follows.Total RNA was isolated from frozen cell pellets using RNAqueous®-4PCRkit (Ambion cat. No. AM1914) used according to manufacturer'sinstructions. Heavy chain V-region mRNA was amplified using a set of sixdegenerate primer pools (HA to HF) and light chain V-region mRNA wasamplified using a set of eight degenerate primer pools, seven for the κcluster (KA to KG) and one for the λ cluster (LA). cDNA for variableregions was prepared from mRNA using RT-PCR. cDNA synthesis was carriedout using constant-region-specific primers, followed by PCR using poolsof degenerate primers for 5′ murine signal sequences and primers to 3′constant regions for each of IgGVH, IgκVL and IgλVL. (Jones et al.,1991, Rapid PCR cloning of full-length mouse immunoglobulin variableregions, Bio/Technology 9:88-89). Heavy and light chain variable regionPCR products were then cloned into a “TA” cloning vector (p-GEM-T easy,Promega cat. no A 1360) and sequenced using standard procedures. SeeFIGS. 2E-F (MAb 8), 2G-H (MAb 13), 2I-J (Mab 16), and 2K-L (Mab 19).

Example 2 Binding Affinity of Anti-hPG Antibodies A. Relative Affinity

Relative affinity of exemplary monoclonal antibodies was measured usingthe ELISA method described above, in which wells were coated with apeptide solution containing 50 ng progastrin and then incubated in thepresence of increasing concentrations of each monoclonal antibody asfollows:

Monoclonal antibody Concentration range tested N-terminal anti-hPG0.001-1 μg/mL monoclonal antibodies MAbs 1-4 N-terminal anti-hPG 0.1-100ng/mL monoclonal antibodies MAbs 3, 15-20 C-terminal anti-hPG 0.03-30ng/mL monoclonal antibodies MAbs 5-14, 21-23

FIG. 3A shows the relative affinity of four anti-hPG monoclonalantibodies, MAbs 1-4 tested at concentrations of 1 ng/mL to 1 μg/mL.FIG. 3B shows the relative affinity of N-terminal anti-hPG monoclonalantibodies MAbs 3 and 15-20 tested at antibody concentrations of 0.1-100ng/mL. FIG. 3C shows the relative affinity of C-terminal anti-hPGmonoclonal antibodies MAbs 5-14 and 21-23 tested at antibodyconcentrations of 0.03-30 ng/mL.

B. Affinity Constant Measurements

To provide a more absolute quantification of affinity for the monoclonalantibodies, affinity constants were measured using the Proteon Technique(BioRad), according to Nahshol et al., 2008, Analytical Biochemistry383:52-60, hereby incorporated by reference in its entirety. Briefly, ananti-mouse IgG antibody (50 μg/ml) was first coated on a sensor chip,making sure that the signal detected by the chip after injection of theantibody falls between 10,000 and 11,500 RU (response units). The murinemonoclonal antibody to be tested was then injected (typicalconcentrations: 30 μg/ml). Sufficient binding of the antibody to betested was determined based on a further signal of at least 500 RU onthe sensor chip. A time-course of interaction between monoclonalanti-progastrin antibodies and progastrin was then measured by injectingvarying concentrations of progastrin, for example 200, 100, 50, 25, and12.5 nM, and the level of association detected. Typically, severalchannels are available to test multiple antibodies in parallel in asingle experiment, making it possible to assay binding of testantibodies at varying concentrations of PG in parallel. Typically, onechannel is reserved for a murine monoclonal antibody that is notspecific to PG, as a control for non-specific binding, and anotherchannel is injected with dilution buffer alone as a baseline for thebackground signal. Generally, no binding is detectable in the channelsinjected with non-specific murine antibody. Antibodies displaying a highlevel of association in this setting, which may result in saturation ofthe trapped monoclonal antibody by progastrin, can be tested againstlower progastrin concentrations (50, 25, 12.5, 6.25 and 3.125 nM),allowing for a more refined measurement.

Affinity constants (KD) were calculated as the ratio between thedissociation constant (kd) and the association constant (ka).Experimental values were validated by analyzing the statisticallyrelevant similarity between experimental curves based on bindingmeasurements and theoretical profiles. The mathematical model used inthe Proteon experiments to select whether experimental curves fit withthe theoretical model was the Langmuir model, based on a 1:1 interactionbetween progastrin molecules and anti-progastrin monoclonal antibodies.

TABLE 6 Affinity constant Monoclonal Antibody measured KD (M) Anti-hPGMAb 1 2.5 μM (2.5 × 10⁻⁶M) Anti-hPG MAb 2 185 nM (1.85 × 10⁻⁷M) Anti-hPGMAb 3 6.4 nM (6.4 × 10⁻⁹M) Anti-hPG MAb 4 3.5 nM (3.5 × 10⁻⁹M) Anti-hPGMAb 5 13 pM (1.30 × 10⁻¹¹M) Anti-hPG MAb 6 0.6 nM (6.38 × 10⁻¹⁰M)Anti-hPG MAb 7 58 pM (5.84 × 10⁻¹¹M) Anti-hPG MAb 8 0.1 nM (1.08 ×10⁻¹⁰M) Anti-hPG MAb 10 3.6 nM (3.62 × 10⁻⁹M) Anti-hPG MAb 11 0.3 nM(3.12 × 10⁻¹⁰M) Anti-hPG MAb 12 0.4 nM (4.43 × 10⁻¹⁰M) Anti-hPG MAb 130.6 nM (6.12 × 10⁻¹⁰M) Anti-hPG MAb 14 6.8 pM (6.86 × 10⁻¹²M) Anti-hPGMAb 15 0.2 nM (2.11 × 10⁻¹⁰M) Anti-hPG MAb 16 0.2 nM (2.78 × 10⁻¹⁰M)Anti-hPG MAb 17 8.3 nM (8.29 × 10⁻⁹M) Anti-hPG MAb 18 1.2 nM (1.24 ×10⁻⁹M) Anti-hPG MAb 19 0.7 nM (7.79 × 10⁻¹⁰M) Anti-hPG MAb 20 0.2 nM(2.47 × 10⁻¹⁰M) Anti-hPG MAb 21 3.9 nM (3.90 × 10⁻⁹M) Anti-hPG MAb 22 5nM (4.94 × 10⁻⁹M) Anti-hPG MAb 23 0.4 μM (3.99 × 10⁻⁷M)

Example 3 Aggregation of Anti-hPG Antibodies

Aggregation of antibodies can reduce therapeutic efficacy by reducingthe amount of antibody available to bind the target protein. Antibodieswith very low levels of aggregation are preferable for therapeuticapplications. Each batch of antibody will vary compared to other batchesof the same monoclonal antibody. It is generally desirable to usebatches with low aggregation. To quantify antibody aggregation, antibodysolutions were placed in a spectrofluorimeter (Photon TechnologyInternational), and irradiated at 280 nm. Diffusion was measured at 280nm, while emission due to aromatic amino acids (mostly tryptophans) wasmeasured at 330 nm. The level of aggregation was then quantified bycalculating the ratio between OD measurement values at 280 nm versus 330nm, as described in Nominé et al., 2003, Biochemistry 42: 4909-4917. Ahigher 280/330 nm ratio indicates a higher amount of aggregation. Theconcentration of antibody used was 15 μg/ml, 5 to 15 times above thatused for in vitro treatments.

Results are shown in Table 7 below and FIG. 4.

TABLE 7 Sample 280/330 nm Ratio Bovine Serum Albumin (n = 1) 6.4Anti-hPG MAb 1 (n = 1) 15.42 Anti-hPG MAb 2 (n = 2) 9.81 Anti-hPG MAb 3(n = 2) 3.08 Anti-hPG MAb 4 (n = 2) 1.94

Example 4 Binding Specificity of Anti-hPG Monoclonal Antibodies

PG is only one of the peptide products of the gastrin gene. Othergastrin gene products have roles in normal homeostasis, but it is PG'srole in CRC that makes it a useful target for therapeutic and diagnosticpurposes. The monoclonal antibodies described herein are specific tofull length progastrin over all other peptides resulting from theexpression and processing of the gastrin gene, such as glycine-extended(G17-gly), amidated (Gastrin17) gastrins, and the C-terminal flankingpeptide (CTFP). These peptides are present in the circulation. Bindingspecificity of anti-hPG monoclonal antibodies was determined by assayingthe antibodies against human progastrin and other gastrin gene-derivedpeptides, using the ELISA assay described above in Example 1.

Specifically, wells coated with a solution containing one of thefollowing peptides at the indicated amounts: progastrin, Keyhole Limpethemocyanin (KLH), amidated gastrin-17 (G17), glycine-extended gastrin 17(Ggly), or C-terminal Flanking Peptide (CTFP):

Screening peptides Test quantity Recombinant Progastrin 50 ng (expt 1)25 ng (expt 2) Human amidated gastrin I (G-17) 50 and 250 ngGlycine-extended gastrin 17 (G17-Gly) 50 and 250 ng KLH 50 and 250 ngC-terminal flanking peptide (CTFP) 50 and 250 ng

In a first experiment, 3 ng/ml (0.3 ng) of anti-hPG MAb 3 and 1 μg/ml(0.1 μg) each of anti-hPG MAbs 1, 2, and 4 were used. See FIG. 5A. In asecond experiment, the binding specificity of anti-hPG MAbs 5 to 14 and21-23 were tested at 0.3 ng/mL, see FIG. 5B, and the binding specificityof anti-hPG MAbs 3, 15-20 were tested at 1 ng/mL, see FIG. 5C.

Antibodies displayed a weak reaction to high quantities of KLH which wascoupled to the antigenic peptide used in some of the immunogens toimmunize the mice in Example 1 above. There was no detectable effect ofBSA on PG binding of any antibodies, including those raised againstimmunogens that included BSA as a carrier.

All antibodies displayed high specificity for binding to full length hPG(tested at 50 and then 25 ng) as compared to other gastrin-gene derivedpeptides, such as amidated gastrin-17, glycine extended gastrin 17, andthe C-terminal flanking peptide, a 5 amino acid peptide that is cleavedfrom progastrin during the normal processing of the polypeptide to formgastrin. The exemplary antibodies showed no detectable binding (signalabove “PBS alone” background) to any the gastrin-gene derived peptidesother than hPG.

Example 5 Competition Assay

The ability of an anti-hPG monoclonal antibody to compete with ananti-hPG polyclonal antibody for binding to progastrin was determinedusing an ELISA with a “capture” anti-hPG antibody and a “detection”anti-hPG antibody. Anti-hPG MAb 3 was assayed as follows: 96-well plateswere pre-coated with C-terminal progastrin polyclonal antibodies raisedagainst a peptide consisting of residues 71 to 80 of hPG. The plateswere then incubated with 100 μM progastrin was incubated, followed byaddition of 10 μg/ml biotinylated N-terminal anti-hPG polyclonalantibody, raised against a peptide consisting of the amino acid sequenceof SEQ ID NO:25, and increasing concentrations of anti-hPG MAb 3monoclonal antibody. Binding of the biotinylated N-terminal anti-hPGpolyclonal antibody was detected by incubating the plates withstreptavidin-HRP followed by OPD, according to standard protocols.Binding was measured by quantifying luminescence.

Results show that increasing concentrations (μg/ml) of Anti-hPG MAb 3decrease the ability of polyclonal anti-hPG antibodies to bind toprogastrin, showing that the monoclonal antibody competes with thepolyclonal antibody. See, FIG. 6.

Example 6 Epitope Mapping

The specific epitopes bound by exemplary monoclonal antibodies weremapped using the SPOT technique and alanine scanning, as described inLaune, D., et al., 2002, J. Immunol. Methods 267:53-70 and Laune, D.,1997, J. Biol. Chem. 272:30937-30944, respectively. In the SPOTtechnique, 15 amino acid peptide sequences spanning a putative epitopeare generated and spotted onto a nitrocellulose membrane which is thenprobed with the test antibody to determine the minimal epitope sequencerecognized by the antibody. Alanine scanning is used to determineresidues within an epitope that are critical for antibody binding: eachresidue within a putative epitope is mutated one by one to an alanine,and the alanine-containing peptides are then probed with the testantibody.

Families of epitope were identified for exemplary antibodies of thepresent disclosure. For N-terminal anti-hPG monoclonal antibodies MAbs1-4 and 15-20, epitopes comprise at least the following sequences: DAPLG(SEQ ID NO:28), PDAPLG (SEQ ID NO:29), PRSQQPD (SEQ ID NO:30), WKPRSQQPD(SEQ ID NO:31), or WKPRSQQPDAPLG (SEQ ID NO:32), as shown in Table 8below.

TABLE 8 PG peptide antigen: MAb SWKPRSQQPDAPLG SEQ ID NO MAb 2 WKPRSQQPDAPLG 32 MAb 4  WKPRSQQPDAPLG 32 MAb 1         PDAPLG 29 MAb 3         DAPLG 28 MAb 17  WKPRSQQPD 31 MAb 18  WKPRSQQPD 31 MAb 19 WKPRSQQPD 31 MAb 20  WKPRSQQPD 31 MAb 15    PRSQQPD 30 MAb 16   PRSQQPD 30

For C-terminal anti-hPG monoclonal antibodies MAbs 5-7, 9-12, 14 and21-23, epitopes comprise at least the following sequences: FGRR (SEQ IDNO:33), MDFGR (SEQ ID NO:34), AEDEN (SEQ ID NO:35), and GWMDFGRR (SEQ IDNO:36), as shown in Table 9 below.

TABLE 9 PG peptide antigen: MAb QGPWLEEEEEAYGWMDFGRRSAEDEN SEQ ID NOMAb 14             GWMDFGRR 36 MAb 11               MDFGR 34 MAb 5                FGRR 33 MAb 6                 FGRR 33 MAb 7                FGRR 33 MAb 9                 FGRR 33 MAb 10                FGRR..E 33 MAb 12                 FGRR 33 MAb 23                     AEDEN 35

Example 7 Neutralizing Activity of Anti-hPG Antibodies on Cancer CellLines (A) Neutralizing Activity of Anti-hPG Monoclonal Antibodies

Anti-hPG monoclonal antibodies decrease cell survival in representativecolorectal cancer cell lines. Suitable colorectal cancer cell lines areknown in the art. For example, HCT116, LS174T, SW480, and SW620 are celllines commonly used to study colon cancer, which produce and secreteprogastrin. Monoclonal antibodies to PG were tested for their ability toinhibit proliferation in these different cell lines. Survival of cellsfrom each HCT116, LST174T, SW480, and SW620 was tested using differentanti-hPG monoclonal antibodies.

For each experiment, 50,000 cells were seeded into 6-well plates inmedium containing fetal calf serum and incubated for 8 hours. Cells wereserum-starved overnight, and starting at 24 hours after seeding (time“T0”), cells were treated in duplicates every 12 h for 48 hours, in theabsence of fetal calf serum, with 1 μg/ml of monoclonal controlantibodies (mouse anti-human IgG1, Calbiochem, Ref #411451) (CT mAb), orwith 1 μg/ml anti-hPG MAb 1-4 as indicated. Upon further quantitation ofmonoclonal antibodies, antibodies were determined to have been used atapproximately 3 to 5 μl. The number of cells at T0 was counted in acontrol well, for each experiment. For HCT116 cells, experiments wereconducted in the presence of 0.5% fetal calf serum. 48 h after the startof the treatment, the number of surviving cells in each well was countedthree times in a blinded experiment. Reduction in CRC cell proliferationor survival was determined by calculating surviving anti-hPG MAB-treatedcells as a percentage of control MAb-treated cells. Cell counts at thestart of treatment (T0) were subtracted from test and control cellcounts measured at 48 hours. Specifically, the number of live cells inboth control and anti-hPG MAb treated wells was counted at 48 hours,then the difference between each cell count and the cell countdetermined at T0, was calculated. The resulting number of anti-hPGMAB-treated cells was then expressed as a percentage of the number ofcontrol MAb-treated cells.

FIG. 7A-C show the effect of anti-hPG MAb 3 and anti-hPG MAb 4 on thesurvival of SW480 cells, LS174T cells, and HCT116 cells fromrepresentative experiments. Results are mean+/−S.E. from 4 wells comingfrom two independent experiments. Treatment with anti-hPG monoclonalantibodies significantly reduced cell number as compared to treatmentwith control antibody. Statistical significance was determined using aStudent's T-test: *=p<0.05, **=p<0.01, and ***=p<0.001. In each cellline, anti-hPG antibodies reduced cell survival. In one cell line,LST174T, cell numbers at the end of 48 hours of treatment with anti-hPGantibodies were lower than the cell numbers at the start of theexperiment, suggesting that the antibodies caused cells to die, inaddition to inhibiting cell proliferation.

Table 10 shows the percent surviving SW480 colorectal cancer cellstreated with each of four monoclonal anti-hPG antibodies as compared toa control monoclonal antibody (mouse anti-human IgG1, Calbiochem, Ref#411451) (CT mAb).

TABLE 10 p (Treated vs Control) SW480 % of Mann Whitney, (T0 = 26 667)Cell numbers -T0 control two-tailed Control mAb 36050 +/− 3228 Anti-hPGMAb 1 30425 +/− 3098 84.4 0.3556 Anti-hPG MAb 2 28925 +/− 2757 80.20.0476 Anti-hPG MAb 3  6050 +/− 1788 16.8 <0.0001 Anti-hPG MAb 4 17560+/− 3439 48.7 0.0002As compared to control, treatment with anti-hPG monoclonal antibodiesreduced survival of cancer cells by 83.2% (Anti-hPG MAb 3), 51.3%(Anti-hPG MAb 4), 19.8% (Anti-hPG MAb 2), and 15.6% (Anti-hPG MAb 1).

The tables below show the percent surviving LS174T and HCT-116 cellstreated with anti-hPG MAb 3 and 4 as compared to the control monoclonalantibody. The data are represented graphically in the correspondingpanels of FIG. 7.

TABLE 11 P (Treated vs Control) HCT-116 % of Mann Whitney, (T0 = 42 750)Cell numbers -T0 control Two-tailed Control MAb 151 250 +/− 9071  MAb362 750 +/− 9194 41.5% <0.0001 MAb4 82 250 +/− 7435 54.4% 0.0001

TABLE 12 P (Treated vs Control) LS 174T % of Mann Whitney, (T0 = 51 666)Cell numbers -T0 control Two-tailed Control MAb 85 334 +/− 7520  MAb3−6666 +/− 5000 −8% 0.0084 MAb4 +8334 +/− 2500  7% 0.0085

Under in vitro assay conditions, complete inhibition of cell growth isnot expected. In cell culture, progastrin is continually secreted bycancer cells and accumulates in the cell culture medium. Progastrinlevels are expected to increase over time more so than would occur incirculation in the body, increasing the ratio of target protein toantibody and diluting the neutralizing effect of the antibodies. Thus,the neutralizing effect observed with the antibodies in vitro isexpected to be stronger in vivo, where progastrin secreted by tumorcells is carried away in the blood stream, lessening its accumulation insitu.

Inhibition of cell proliferation by anti-hPG MAbs 5 to 23 was determinedin one or more of CRC cell lines SW620, HCT116, and LS47T. Assays wereperformed in E-well plates as described above using 5 μg/ml control ortest (anti-hPG) monoclonal antibodies. 50,000 cells were seeded per wellfor HCT116 and LS174T, and 100,000 for SW620 cells. The tables belowprovide percent of surviving treated cells relative the controltreatment from representative experiments. Average results are graphedin FIG. 7G-I for cell lines SW620, LS174T, and HCT-116 respectively.

TABLE 13 P (Treated vs Control) % of Mann Whitney, Cell numbers -T0control Two-tailed Experiment 1 SW 620 (T0 = 103 067) Control MAb  82100 +/− 15489 — MAb 5 54 511 +/− 8292 66% <0.0001 MAb 6  44367 +/− 932154% <0.0001 MAb 7  49279 +/− 8009 60% <0.0001 MAb 8  32673 +/− 4680 40%<0.0001 MAb 9  73283 +/− 3835 89% 0.1305 MAb 10  70178 +/− 4173 85%0.0618 Experiment 2 SW 620 (T0 = 118 553) Control MAb 81 347 +/− 6062MAb 11 46 974 +/− 7422 58% 0.0003 MAb 12  52 980 +/− 10529 65% 0.0002MAb 13 38 933 +/− 5284 48% 0.0003 MAb 14 83 767 +/− 9484 103%  0.21 MAb21 59 497 +/− 2828 73% 0.0002 MAb 22 64 227 +/− 7123 79% 0.0013 MAb 2383 914 +/− 5629 103%  0.82 Experiment 3 SW 620 (T0 = 116 283) ControlMAb  101 333 +/− 17 626 — MAb 15 66 052 +/− 7739 65% <0.0001 MAb 16 58883 +/− 9950 58% <0.0001 MAb 17 76 688 +/− 5578 75.5%  0.0014 MAb 18  75874 +/− 10129 75% 0.0005 MAb 19  70 242 +/− 10 851 69% <0.0001 MAb 20 66470 +/− 7557 66% <0.0001

TABLE 14 P (Treated vs Control) % of Mann Whitney, Cell numbers -T0control Two-tailed Experiment 1 LS 174T (T0 = 60 944) Control MAb 107956 +/− 5859  MAb 13  62 341 +/− 10 683 58% 0.0003 MAb 16 65 389 +/−8185  61% 0.0002 Experiment 2 LS 174T (T0 = 86 389) Control Mab 241 711+/− 11 620 MAb 14 246 444 +/− 19563  102%  ns MAb 19 204 433 +/− 8946 84.5%  0.0005 Experiment 3 LS 174T (T0 = 79 667) Control MAb 135 800 +/−18 338 MAb 8  57 333 +/− 12657 42% <0.001

TABLE 15 P (Treated vs Control) % of Mann Whitney, Cell numbers -T0control Two-tailed Experiment 1 HCT-116 (T0 = 49 286) Control MAb 78 214+/− 6230 MAb 13 28 805 +/− 3477 36% <0.0001 MAb 16 56 484 +/− 8333 72%<0.0001 MAb 19 68 945 +/− 8795 88% 0.0302 Experiment 2 HCT-116 (T0 = 60944) Control MAb 122 456 +/− 1697  MAb 8  75 867 +/− 15627 62% <0.0001MAb 16  87011 +/− 5091 71% <0.0001

(B) Neutralizing Activity of Anti-hPG Polyclonal Antibodies

Assays were conducted as described above, with the followingmodifications. An N-terminal anti-hPG polyclonal antibody as describedin Example 5 was used. As a control, 3 μg/ml of polyclonal (PolyclonalRabbit anti-human IgG, Affinity BioReagents, Ref #SA1-600) (CT pAB) wasused. For anti-PG treatments, 3 μg/ml anti-hPG polyclonal antibodieswere used for all cell lines. SW480 and LS174T were treated with controlor N-terminal anti-hPG polyclonal antibodies for 24 to 48 h in DMEMwithout FCS, while HCT116 were treated with anti-hPG polyclonalantibodies for 48 h in DMEM with 0.5% FCS. Surviving cells were thentrypsinized and counted, in comparison with cells treated with anequivalent concentration of control (anti human IgG) polyclonalantibody.

Results of representative experiments are shown in the tables below andFIG. 7D-F. Treatment with anti-hPG polyclonal antibodies significantlyreduced cell number as compared to treatment with control antibody.Statistical significance was determined using a Student's T-test:*=p<0.05, **=p<0.01, and ***=p<0.001. Cell numbers are expressedrelative to the number of cells in culture at the start of theexperiment (T0). For each experiment, the cells in each of 4 wells werecounted three times. As with anti-hPG monoclonal antibodies, colorectalcancer cell proliferation is inhibited by anti-hPG polyclonalantibodies, demonstrating that anti-tumor effects seen with polyclonalantibodies to progastrin are reasonably predictive of monoclonalantibody activity in blocking progastrin's effect on cancer cells.

TABLE 16 P (Treated vs Control) % of Mann Whitney, Cell numbers -T0control Two-tailed Experiment 1 SW 480 (T0 = 26 667) Control PAb  37 580+/− 4233 PG PAb   7833 +/− 3660 21% 0.0001 Experiment 2 HCT-116 (T0 = 58750) Control PAb 105 350 +/− 8660 PG PAb   7833 +/− 3660 21% <0.05Experiment 3 LS174T (T0 = 112 500) Control PAb  207 500 +/− 10 000 PGPAb 102 500 +/− 5000 49.5%  <0.01

Example 8 Neutralizing Effect of Anti-hPG Monoclonal Antibodies isEliminated when Antibodies are Pre-Incubated with Purified hPG

To demonstrate that the neutralizing effect of anti-hPG monoclonalantibodies is mediated by binding to hPG, LS174T cells were cultured inthe presence of an exemplary antibody—anti-hPG MAb 8—that had beenpre-incubated with and without hPG. As positive and negative controls,cells were cultured with hPG alone, a control antibody alone, and thecontrol antibody pre-incubated with hPG.

Specifically, 33.3 nM (5 μg/mL) anti-hPG MAb 8 was pre-incubated for 1hour at room temperature with 20-fold molar excess recombinant hPG, or667 nM (6.67 μg/mL). Recombinant human progastrin, prepared as describedin Example 1, was used. In parallel, 33.3 nM (5 μg/mL) of murineanti-human IgG1 (General BioScience, reference no. AB23420) wassimilarly pre-incubated with and without hPG.

5000 LS174T cells were seeded into each well in 96-well plates in mediumcontaining 10% Fetal Calf Serum and incubated for 8 hours, after whichthe cells were switched to serum-free medium and grown for another 12hours. After growth in serum-free medium for 12 hours, cells weretreated with one of the following every twelve hours: control antibody,control antibody pre-incubated with hPG, anti-hPG MAb 8 alone, anti-hPGMAb 8 pre-incubated with hPG, and hPG alone. 48 hours after the firsttreatment, remaining viable cells were quantified by incubating plateswith Promega CellTiter 96 Aqueous One Solution and recording theabsorbance at 490 nM. The absorbance measured for cells treated with thecontrol monoclonal antibody (“control MAb”) was set to 100%, and allother experimental conditions measured against the absorbance of cellstreated with control MAb. Results are shown in the Table below and FIG.8.

TABLE 17 p (Treated vs Control) % of Mann Whitney, Absorbance controltwo-tailed PG treatment alone 0.244 +/− 0.088 132.5% 0.099 (n.s.)Control MAb 0.184 +/− 0.084  100% N.A. Anti-hPG MAb 8 0.057 +/− 0.06   31% 0.001 Control MAb + hPG 0.321 +/− 0.079 174.5% 0.002 Anti-hPGMAb8 + hPG 0.271 +/− 0.076 147.6% 0.0229

Addition of, or incubation of antibodies with, hPG increases the numberof live cells in culture. In contrast, treatment of the cells withanti-hPG MAb 8 alone causes a significant reduction in the number ofviable cells. Thus, the ability of anti-hPG monoclonal antibodies toneutralize PG activity is abolished by the addition of hPG, which isthought to bind to and saturate the antibody. This result confirms thespecificity of the neutralizing activity of anti-hPG monoclonalantibodies.

Example 9 In Vivo Anti-Tumor Activity of Anti-hPG Antibodies

A number of experimental in vivo models have been developed for thestudy of colorectal cancer. Mouse xenograft studies, in which tumortissue or cells from human cancer cell lines are transplanted into animmunodeficient (so-called “nude”) mouse, have been developed. PocardM., et al., In vivo (1996) 10(5): 463-469. Several transgenic mousemodels have also been developed. Murine models include heterozygousmutations in the adenomatous polyposis coli (APC) gene, such asApc^(Min), Apc1638N, Apc716, or ApcΔ14. The APC tumor suppressor geneencodes a cytosolic protein, APC, which, when intact, binds to andsequesters β-catenin in the cytosol within a multi-protein complextargeting β-catenin to the proteasome for degradation, therebypreventing β-catenin from activating the transcription factor Tcf-4 inthe nucleus. Heyer et al., Oncogene 18:5325-5333 (1999). APCΔ14 micecarry a heterozygous deletion of exon 14 within the adenomateouspolyposis coli (APC) gene. Similar to what occurs in more than 70% ofpatients with sporadic colorectal cancer, somatic loss of heterozygocity(LOH) in the second Apc allele occurs in intestinal cells, leading to aconstitutive activation of the β-catenin/Tcf-4 transcriptional complex,and to the spontaneous development of intestinal tumors in theseanimals. The molecular origin of these adenomas and carcinomas, as wellas tumor morphology (including vascularization, inflammatory responseand presence of immune cells) with much greater similarity to that ofhuman tumors compared with mouse xenograft models, make APCΔ14 a highlyrelevant model for human colorectal cancer therapy studies. Colnot etal., 2004, Lab. Invest. 84:1619-1630. Other transgenic mouse models arebased on mutations in genes such as MSH2, MSH6, CDX2, K-RAS, as well aslines combining mutations in APC with mutations in other oncogenes.Heyer et al., 1999, Oncogene 18:5325-5333, Janssen K P et al., 2002,Gastroenterology 123: 492-504. These models are widely used to studycolorectal cancer and test hypotheses regarding the treatment ofcolorectal cancer.

Transgenic mice carrying a heterozygous deletion of exon 14 with theadenomatous polyposis coli gene (APCΔ14) serve as a model for colorectalcancer, developing tumors similar to those found in human colon cancers.In a first experiment, APCΔ14 mice were treated with a preparationcontaining equal amounts of anti-hPG polyclonal antibodies raisedagainst (1) a peptide corresponding to SEQ ID NO:25 and (2) a C-terminalpeptide as described in Hollande et al., WO 07/135,542. 3.5 month oldmice were treated for 5 weeks with either control polyclonal antibody oranti-hPG polyclonal antibody (two mice per treatment). Antibodies wereadministered by intra-peritoneal injection twice a week at a dose of 10mg/kg (150 μl injection volume). At the end of the treatment, mice weresacrificed and the intestines were washed with PBS, dissected fordigital imaging and fixed in 4% para-formaldehyde forimmuno-histochemical analysis. Tumor number and images of colorectaltissue were recorded.

Morphological assessment of intestinal tissue showed that anti-hPGantibodies did not affect the renewal of healthy murine intestinalepithelium. Tumors were counted in the treatment and control groups. Thetotal number of tumors for the mice treated with control antibodies was27, as compared to 4 in the mice treated with anti-hPG antibodies. Thus,anti-hPG antibodies reduce tumor count by more than 6.5 fold as comparedto control antibodies while not affected the renewal of healthyepithelium in the mouse intestine.

In a second experiment, effect of anti-hPG antibodies in APCΔ14 mice andnormal (control) mice (C57BL6J) was examined. 4-month old mice weretreated twice a week for 6 weeks with either control polyclonalantibody—a rabbit anti-human IgG antiserum (Jackson ImmunoResearch(reference no. 309-005-0089)—or anti-hPG polyclonal antibody, byintra-peritoneal injection twice a week at a dose of 9 mg/kg (150injection volume). After six weeks of treatment, two hours beforesacrifice, mice were injected with Bromo-deoxy-uridine (BrdU) (2 mg permouse, intraperitoneal injection). 6 APCΔ14 mice were treated withanti-hPG polyclonal and 4 with control polyclonal antibodies. Controland anti-hPG antibodies were administered to 6 normal (C57BL6J) miceeach. No intestinal abnormalities were detected in any of the mice fromeither group, further demonstrating the safety and lack of adverseeffect of anti-hPG antibodies on normal intestinal tissue.

Tumor numbers and size (height and length) was examined in treatedversus control groups of APCΔ14 mice. Tumor size was determined bymeasurements of images taken from intestines of each animal, prepared asfollows. Intestines were rinsed as described above, dissectedlongitudinally, embedded in paraffin, and processed forimmuno-histochemistry using the “Swiss roll” technique. Measurements oftumor length and height were performed using Image J software.

Results are shown in Table 18 and FIGS. 8A and B. Results for tumor sizeshow a statistically significant difference between control-treated andanti-hPG-treated groups. Statistical significance was determined using aMann Whitney test: *=p<0.05, **=p<0.01, and ***=p<0.001. Mice treatedwith control antibody exhibited a total of 125 tumors, with 31.25 tumorson average per mouse. Anti-hPG treated mice exhibited 46 tumors or anaverage of 7.6 tumors per mouse. This difference is statisticallysignificant (Maim-Whitney test, P=0.0095) showing that anti-hPGantibodies significantly reduces the number of colorectal cancer tumorsin vivo.

TABLE 18 Treatment Number of (no. of mice) tumors per mouse Control PAb(4) 23 48 28 26 Anti-hPG PAb (6) 2 16 15 9 2 2

Anti-hPG antibodies significantly reduce colorectal cancer tumors invivo as measured by reduction in both tumor number and tumor size in amurine model of colorectal cancer, without any apparent adverse affecton normal colorectal epithelium. Thus, treatment of colorectal cancertumors with anti-hPG provides a therapeutic benefit in vivo.

Example 10 Design of Humanized Anti-hPG Antibodies

Humanized antibodies were designed “in silico” from murine anti-hPGmonoclonal antibodies MAbs 3, 4, 8, 13, 16, and 19. Design of humanizedantibodies was carried out carried out according to the methodologydescribed in Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77; Lefrancet al., 2009, Nucl. Acids Res. 37: D1006-1012; Lefranc, 2008, Mol.Biotechnol. 40: 101-111. For each of the murine monoclonal antibodies,corresponding humanized V_(H) and V_(L) peptide sequences weredetermined by: identifying the CDR and framework regions in the murinesequences using the IMGT-ONTOLOGY database (Duroux et al., 2008,Biochimie, 90:570-583; Giudicelli and Lefranc, 1999, Bioinformatics, 15:1047-1054) and IMGT® databases and tools (Ehrenmann et al., 2010, Nucl.Acids. Res., 38: D301-307; Brochet et al., 2008, Nucl. Acids. Res., 36:W503-508) followed by identification of the amino acid sequence of theclosest human framework region sequences in the IMGT/GENE-DB (Giudicelliet al., 2005, Nucl. Acids. Res., 33: D256-261), and grafting of themurine CDR sequences onto the human framework regions. Moreparticularly, nucleotide and amino acid sequences of murine V_(H) andV_(L) domains were analyzed using IMGT/V-QUEST and IMGT/DomainGapAlignto delimit the murine CDRs and framework regions, define CDR lengths andidentify anchor amino acids. Anchor amino acids are residues at position26, 39, 55, 66, 104 and 118 of IMGT “Collier de Perles” that support theCDR1-IMGT, CDR2-IMGT, CDR3-IMGT (Kaas and Lefranc, 2007, CurrentBioinformatics, 2: 21-30; Kaas et al., Brief. Funct. Genomic Proteomic,2007, 6: 253-264). The closest human V (variable) and J (joining) genesto the murine sequences were identified and the most suitable geneschosen. Individual amino acids in the murine framework region weremaintained if they were considered to possibly contribute to thespecificity of the antibody by comparison with known 3D structures (Kaaset al., 2004, Nucl. Acids. Res. 32: 208-210) using IMGT Collier dePerles on two layers.

V_(H) CDRs for MAb 3 were determined to be 8, 8, and 8 amino acids longfor CDR 1, 2, and 3 respectively. The closest germline mouse gene to thesequence of the murine MAb 3 V_(H), IGHV1-5*01, differed at 10 residues,3 of which were mapped to the CDRs and were kept in the design ofhumanized anti-hPG monoclonal antibodies (I29 in CDR1, F56 and S65 inCDR2). In addition, V39, G55, and R66 were considered potentiallyimportant for preserving specificity and were kept in the design. Theclosest human germline gene was IGHV1-3*01 (63.3% identical at the aminoacid level based on IGMT/DomainGapAlign). 27 sequence differences in theframework regions 1 to 3 were found between the murine and humangermline genes. In framework region 4, Threonine-123 and Leucine-124were changed to Leucine-123 and Valine-124, so as to match the humanIGHJ1*01 gene. Overall, the humanized V_(H) for MAb 3 is 87.8% identicalto the variable region for IGHV1-46*03 and 88 of the 91 amino acids inthe four framework regions are identical.

V_(L) CDRs for MAb 3 were determined to be 11, 3, and 9 amino acids longfor CDR 1, 2, and 3 respectively. The closest germline mouse gene to thesequence of the murine MAb 3 V_(L), IGKV1-117*01, differed at a singleresidue mapping to a framework region. The closest human germline genewas IGKV2-30*02 (81% identical at the amino acid level based onIGMT/DomainGapAlign). 14 sequence differences in the framework regions 1to 3 were found between the murine and human germline genes. ResiduesE40 and F68 have been maintained in the projected humanized sequence. Inframework region 4, Leucine-124 was changed to Valine-124, so as tomatch the human IGKJ4*01 gene. Overall, the humanized Vκ sequence forMAb 3 is 93% identical to IGKV2D-29*02 and 87 of the 89 amino acids inthe four framework regions are identical.

Projected V_(H) and Vκ sequences are provided in the table below.

TABLE 19 SEQ ID V NO:  Amino acid sequence chain 21QVQLVQSGAEVKKPGASVKVSCKASGYIFTSYWVHWVRQ hV_(H)3APGQRLEWMGGFYPGNSDSRYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCTRRDSPQYWGQGTLVTVSS 22DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLE  hVκ3WFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFTFGGGTKVEIK

V_(H) CDRs for MAb 4 were determined to be 8, 8, and 11 amino acids longfor CDR 1, 2, and 3 respectively. The closest germline mouse gene to thesequence of the murine MAb 4 V_(H), IGHV1-9*01, differed at 11 residues,4 of which were mapped to the CDRs and were kept in the design ofhumanized anti-hPG monoclonal antibodies (S35, S36, and S37 in CDR1, F56in CDR2). In addition, D66 was considered potentially important forpreserving specificity and was kept in the design. The closest humangermline gene was IGHV1-46*03 (64.9% identical at the amino acid levelbased on IGMT/DomainGapAlign). 27 sequence differences in the frameworkregions 1 to 3 were found between the murine and human germline genes.In framework region 4, Alanine-128 was changed to Serine-128, so as tomatch the human IGHJ5*01 gene. Overall, the humanized V_(H) for MAb 4 is91.8% identical to the variable region for IGHV1-46*03 and 90 of the 91amino acids in the four framework regions are identical.

V_(L) CDRs for MAb 4 were determined to be 11, 3, and 9 amino acids longfor CDR 1, 2, and 3 respectively. The closest germline mouse gene to thesequence of the murine MAb 4 V_(L), IGKV1-110*01, differed at 3residues, 2 of which were mapped to CDR1 (Serine-34 and Valine-36) andwere kept in the design of humanized anti-hPG monoclonal antibodies. Theclosest human germline gene was IGKV2D-29*02 (81% identical at the aminoacid level based on IGMT/DomainGapAlign). 11 sequence differences in theframework regions 1 to 3 were found between the murine and humangermline genes. In framework region 4, Serine-120 was changed toGlutamine-120, so as to match the human IGKJ2*01 gene. Overall, thehumanized Vκ sequence for MAb 4 is 92% identical to IGKV2D-29*02 and100% identical for the four framework regions.

Projected V_(H) and Vκ sequences are provided in the table below.

TABLE 20 SEQ ID V NO: Amino acid sequence chain 23QVQLVQSGAEVKKPGASVKVSCKASGYTFSSSWMHWVRQAPG hV_(H)4QGLEWMGIFLPGSGSTDYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCATDGNYDWFAYWGQGTLVTVSS 24DIVMTQTPLSLSVTPGQPASISCKSSQSLVHSSGVTYLYWYL hVκ4QKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCSQSTHVPPTFGQGTKLEIK

V_(H) CDRs for MAb 8 were determined to be 8, 8, and 10 amino acids longfor CDR 1, 2, and 3 respectively. The closest germline mouse gene to thesequence of the murine MAb8 V_(H), IGHV5-9-3*01, differed at 5 residues,4 of which were mapped to the CDRs and were kept in the design ofhumanized anti-hPG monoclonal antibodies. The closest human germlinegenes were IGHV3-21*01 and *02 (80.4% identical at the amino acid levelbased on IGMT/DomainGapAlign). 12 sequence differences in the frameworkregions 1 to 3 were found between the murine and human germline genes.In framework region 4, Serine-123 and Leucine-124 were changed toThreonine-123 and Valine-124, respectively, so as to match the humanIGHJ6*01 gene. Overall, the humanized V_(H) for MAb 8 is 92.8% identicalto the variable region for IGHV3-21*01 and *02 and 100% identical forthe four framework regions. There is a potential N-glycosylation site inmurine V_(H) CDR3 for MAb8.

V_(L) CDRs for MAb 8 were determined to be 11, 3, and 9 amino acids longfor CDR 1, 2, and 3 respectively. The closest germline mouse gene to thesequence of the murine MAb 8 V_(L), IGKV2-109*01, differed at 6residues, 4 of which were mapped to CDR1 and were kept in the design ofhumanized anti-hPG monoclonal antibodies. The closest human germlinegenes were IGKV2-28*01 and IGKV2D-28*01 (75% identical at the amino acidlevel based on IGMT/DomainGapAlign). 12 sequence differences in theframework regions 1 to 3 were found between the murine and humangermline genes. In framework region 4, Alanine-120, Leucine-124, andLeucine 126 were changed to Glycine-120, Valine-124, and Isoleucine-126,respectively, so as to match the human IGKJ4*01 gene. Overall, thehumanized Vκ sequence for MAb 8 is 87% identical to IGKV2-28*01 andIGKV2D-28*01 and 100% identical for the four framework regions.

Projected V_(H) and Vκ sequences are provided in the table below.

TABLE 21 SEQ ID NO:  Amino acid sequence V chain 75EVQLVESGGGLVKPGGSLRLSCAASGFT hV_(H)8a FTTYAMNWVRQAPGKGLEWVSSISSGGTYTYYADSVKGRFTISRDNAKNSLYLQMN SLRAEDTAVYYCATQGNYSLDFWGQGTT VTVSS 76DIVMTQSPLSLPVTPGEPA hVκ8a SISCRSSKSLRHTKGITFLDWYLQKPGQSPQLLIYQMSNRASGVPD RFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGGGTKVEIK 77 EVQLVESGGGLVKPGGSLRLSCAA hV_(H)8bSGFTFTTYAMSWVRQAPGKGLEWVS SISSGGTYTYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCATQGNYSLDFWGQGTTVTVSS 78 DIVMTQSPLSLPVTPGEPASISC hVκ8bRSSKSLRHTKGITFLYWYLQKPG QSPQLLIYQMSNRASGVPD RFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGGGTKVEIK 79 EVQLVESGGGLVKPGGSLRLSCAASGF hV_(H)8bTFTTYAMSWVRQAPGKGLEWVSTISSG GTYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCATQGNYSLDFWGQGTTVTVSS

V_(H) CDRs for MAb 13 were determined to be 8, 8, and 7 amino acids longfor CDR 1, 2, and 3 respectively. The closest germline mouse gene to thesequence of the murine MAb 13 V_(H), IGHV5-6-3*01, differed at 10residues, 5 of which were mapped to the CDRs and were kept in the designof humanized anti-hPG monoclonal antibodies. The closest human germlinegenes were IGHV3-7*01 and *02 (78.6% identical at the amino acid levelbased on IGMT/DomainGapAlign). 13 sequence differences in the frameworkregions 1 to 3 were found between the murine and human germline genes.In framework region 4, Threonine-123 and Leucine-124 were changed toLeucine-123 and Valine-124, respectively, so as to match the humanIGHJ4*01 gene. Overall, the humanized V_(H) for MAb 13 is 91.8%identical to the variable region for IGHV3-7*01 and *02 and 100%identical for the four framework regions.

V_(L) CDRs for MAb 13 were determined to be 11, 3, and 9 amino acidslong for CDR 1, 2, and 3 respectively. The closest germline mouse geneto the sequence of the murine MAb 13 V_(L), IGKV1-135*01, differed at asingle residue located in a framework region. The closest human germlinegenes were IGKV2-30*01 and *02 (81% identical at the amino acid levelbased on IGMT/DomainGapAlign). 13 sequence differences in the frameworkregions 1 to 3 were found between the murine and human germline genes.In framework region 4, Leucine-124 was changed to Valine-124, so as tomatch the human IGKJ4*01 gene. Overall, the humanized Vκ sequence forMAb 13 is 94% identical to IGKV2-30*01 and *02 and 100% identical forthe four framework regions.

Projected V_(H) and Vκ sequences are provided in the table below.

TABLE 22 SEQ ID V NO: Amino acid sequence chain 80EVQLVESGGGLVQPGGSLRLSCAASGFIFSSYGMSWVRQ hV_(H).13aAPGKGLEWVANINTFGDRTYYVDSVKGRFTISRDNAKITSLYLQMNSLRAEDTAVYYCARGTGTYWGQGTLVTVSS 81DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLN  hVκ13aWFQQRPGQSPRRLIYLVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGGGTKVEIK 82EVQLVESGGGLVQPGGSLRLSCAASGFIFSSYGMSWVRQ hV_(H)13bAPGKGLEWVASINTFGDRTYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGTGTYWGQGTLVTVSS 83DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLN hVκ13bWFQQRPGQSPRRLIYLVSKRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGGGTKVEIK

V_(H) CDRs for MAb 16 were determined to be 8, 8, and 10 amino acidslong for CDR 1, 2, and 3 respectively. The closest germline mouse geneto the sequence of the murine MAb 16, V_(H), IGHV1-53*01, differed at 7residues, 2 of which were mapped to the CDRs and were kept in the designof humanized anti-hPG monoclonal antibodies. The closest human germlinegenes were IGHV1-46*01 and *03 (71.4% identical at the amino acid levelbased on IGMT/DomainGapAlign). 25 sequence differences in the frameworkregions 1 to 3 were found between the murine and human germline genes.In framework region 4, Leucine-124 was changed to Valine-124, so as tomatch the human IGHJ6*01 gene. Overall, the humanized V_(H) for MAb 16is 96.9% identical to the variable region for IGHV1-46*01 and *03 and100% identical for the four framework regions.

V_(L) CDRs for MAb 16 were determined to be 11, 3, and 9 amino acidslong for CDR 1, 2, and 3 respectively. The closest germline mouse geneto the sequence of the murine MAb 16, V_(L), IGKV1-135*01, differed at 4residues located in a framework region. The closest human germline geneswere IGKV2-30*01 and *02 (79% identical at the amino acid level based onIGMT/DomainGapAlign), that differed by one amino acid in CDR1. 15sequence differences in the framework regions 1 to 3 were found betweenthe murine and human germline genes. In framework region 4, Glycine-120was changed to Glutamine-120, so as to match the human IGKJ2*01 gene.Overall, the humanized Vκ sequence for MAb 16 is 94% identical toIGKV2-30*01 and *02 and 100% identical for the four framework regions.

Projected V_(H) and Vκ sequences are provided in the table below.

TABLE 23 SEQ ID V NO: Amino acid sequence chain 84QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQ hV_(H)16aAPGQGLEWMGIINPSNGGTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTRGGYYPFDYWGQGTTVTVSS 85DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLN hVκ16aWFQQRPGQSPRRLIYLVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHSPYTFGQGTKLEIK 86QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQ hV_(H)16bAPGQGLEWMGIINPSNGGTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTRGGYYPFDYWGQGTTVTVSS 87DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLY hVκ16bFWQQRPGQSPRRLIYLVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHSPYTFGQGTKLEIK 88QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQ hV_(H)16cAPGQGLEWMGEINPSNGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTRGGYYPFDYWGQGTTVTVSS 89DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLY hVκ16cWFQQRPGQSPRRLIYLVSERDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHSPYTFGQGTKLEIK

V_(H) CDRs for MAb 19 were determined to be 9, 7, and 14 amino acidslong for CDR 1, 2, and 3 respectively. The closest germline mouse geneto the sequence of the murine MAb 19 V_(H), IGHV3-2*01, differed at 5residues, 2 of which were mapped to the CDRs and were kept in the designof humanized anti-hPG monoclonal antibodies. The closest human germlinegene was IGHV4-30-4*01 (72.4% identical at the amino acid level based onIGMT/DomainGapAlign). However, since this gene is polymorphic,IGHV4-31*02 and *03 (71.4% identical at the amino acid level based onIGMT/DomainGapAlign) were selected. 21 sequence differences in theframework regions 1 to 3 were found between the murine and humangermline genes. In framework region 4, Isoleucine-123 was changed toLeucine-123, so as to match the human IGHJ4*01 gene. Overall, thehumanized V_(H) for MAb 19 is 91.9% identical to the variable region forIGHV4-31*02 and *03 and 100% identical for the four framework regions.

V_(L) CDRs for MAb 19 were determined to be 7, 7, and 13 amino acidslong for CDR 1, 2, and 3 respectively. The closest germline mouse geneto the sequence of the murine MAb 19 V_(L), IGLV3*01, differed at 8residues, 5 of which were located in a CDR. The closest human germlinegenes were IGLV4-69*01 and *02 (69.9% identical at the amino acid levelbased on IGMT/DomainGapAlign). 23 sequence differences in the frameworkregions 1 to 3 were found between the murine and human germline genes.In framework region 4, Valine-124 was changed to Leucine-124, so as tomatch the human IGLJ3*01 gene. Alternatively, the IGJK4*01 gene can beused for the framework 4 region. Overall, the humanized Vκ sequence forMAb 19 is 92.4% identical to IGLV4-69*01 and *02 and 100% identical forthe four framework regions.

Projected V_(H) and Vκ sequences are provided in the table below.

TABLE 24 SEQ ID V NO:  Amino acid sequence chain 90QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDYAWSWIRQH V_(H)19aPGKGLEWIGYISFSGYTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREVNYGDSYHFDYWGQGTLVTVSS 91QLVLTQSPSASASLGASVKLTCTLSSQHRTYTIAWHQQQPE Vκ19aKGPRYLMKVKKDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGVGDAIKGQSVFVFGGGTKVEIK 92QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDYAWNWIRQH V_(H)19bPGKGLEWIGYISFSGYTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREVNYGDSYHFDYWGQGTLVTVSS 93QLVLTQSPSASASLGASVKLTCTLSSQHRTYTIEWHQQQPE Vκ19bKGPRYLMKVKKDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGVGDAIKGQSVFVFGGGTKVEIK 94QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDYAWNWIRQH V_(H)19cPGKGLEWIGYISFSGYTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREVNYGDSYHFDYWGQGTLVTVSS 95QLVLTQSPSASASLGASVKLTCTLSSQHRTYTIEWHQQQPE Vκ19cKGPRYLMEVKKDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGVGDAIKGQSVFVFGGGTKVEIK

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

1. A monoclonal antibody that specifically binds human progastrin (hPG).2. The monoclonal antibody of claim 1 which specifically binds anN-terminal region of hPG.
 3. The monoclonal antibody of claim 2 which isobtainable using an immunogen comprising a peptide antigen having anamino acid sequence corresponding to SEQ ID NO:25.
 4. The monoclonalantibody of claim 2 that competes for binding hPG with a referenceanti-hPG monoclonal antibody obtainable from a hybridoma selected fromthe group consisting of 43B9G11, WE5H2G7, 6B5B11C10, 20D2C3G2, 1E9A4A4,1E9D9B6, 1C8D10F5, 1A7C3F11, 1B3B4F11, and 1C11F5E8.
 5. The monoclonalantibody of claim 2 which comprises V_(L) and V_(H) CDRs correspondingin sequence to the V_(L) and V_(H) CDRs of the monoclonal antibodiesobtainable from a hybridoma selected from the group consisting of:43B9G11, WE5H2G7, 6B5B11C10, 20D2C3G2, 1E9A4A4, 1E9D9B6, 1C8D10F5,1A7C3F11, 1B3B4F11, and 1C11F5E8.
 6. The monoclonal antibody of claim 2which comprises V_(L) and V_(H) CDRs having sequences selected from oneof the following groups of V_(L) and V_(H) CDR sequences: (i) V_(H)CDR1.3, V_(H) CDR 2.3, V_(H) CDR 3.3, V_(L) CDR 1.3, V_(L) CDR 2.3, andV_(L) CDR 3.3; (ii) V_(H) CDR 1.4, V_(H) CDR 2.4, V_(H) CDR 3.4, V_(L)CDR 1.4, V_(L) CDR 2.4, and V_(L) CDR 3.4; (iii) V_(H) CDR 1.16, V_(H)CDR 2.16, V_(H) CDR 3.16, V_(L) CDR 1.16, V_(L) CDR 2.16, and, V_(L) CDR3.16; and (iv) V_(H) CDR 1.19, V_(H) CDR 2.19, V_(H) CDR 3.19, V_(L) CDR1.19, V_(L) CDR 2.19, and V_(L) CDR 3.19.
 7. The monoclonal antibody ofclaim 1, which is humanized.
 8. The monoclonal antibody of claim 7 whichcomprises V_(H) and V_(L) chains having sequences selected from one ofthe following groups of V_(H) and V_(L) sequences: (i) hV_(H) 3 (SEQ IDNO:21) and hV_(L) 3 (SEQ ID NO:22); (ii) hV_(H) 4 (SEQ ID NO:23) andhV_(L) 4 (SEQ ID NO:24); (iii) hV_(H) 16a (SEQ ID NO:84) and hV_(L) 16a(SEQ ID NO:85); (iv) hV_(H) 16b (SEQ ID NO:86) and hV_(L) 16b (SEQ IDNO:87); (v) hV_(H) 16c (SEQ ID NO:88) and hV_(L) 16c (SEQ ID NO:89);(vi) hV_(H) 19a (SEQ ID NO:90) and hV_(L) 19a (SEQ ID NO:91); (vii)hV_(H) 19b (SEQ ID NO:92) and hV_(L) 19b (SEQ ID NO:93); and (viii)hV_(H) 19c (SEQ ID NO:94) and hV_(L) 19c (SEQ ID NO:95).
 9. Themonoclonal antibody of claim 1 which specifically binds a C-terminalregion of hPG.
 10. The monoclonal antibody of claim 9 which isobtainable using an immunogen comprising a peptide antigen having anamino acid sequence corresponding to SEQ ID NO:27.
 11. The monoclonalantibody of claim 9 which competes for binding hPG with a referenceanti-hPG monoclonal antibody obtainable from a hybridoma selected fromthe group consisting of 1B4A11D11, 1B6A11F2, 1B11E4B11, 1C10D3B9,1D8F5B3, 1E1C7B4, 2B4C8C8, 2B11E6G4, 2C6C3C7, 2H9F4B7, 1F11F5E10,1F11F5G9, and 1A11F2C9.
 12. The monoclonal antibody of claim 9 whichcomprises V_(L) and V_(H) CDRs corresponding in sequence to the V_(L)and V_(H) CDRs of the monoclonal antibodies obtainable from a hybridomaselected from the group consisting of: 1B4A11D11, 1B6A11F2, 1B11E4B11,1C10D3B9, 1D8F5B3, 1E1C7B4, 2B4C8C8, 2B11E6G4, 2C6C3C7, 2H9F4B7,1F11F5E10, 1F11F5G9, and 1A11F2C9.
 13. The monoclonal antibody of claim9 which comprises V_(H) and V_(L) CDRs having sequences selected fromone of the following groups of V_(H) and V_(L) CDR sequences: (i) V_(H)CDR 1.8, V_(H) CDR 2.8, V_(H) CDR 3.8, V_(L) CDR 1.8, V_(L) CDR 2.8, andV_(L) CDR 3.8; and (ii) V_(H) CDR 1.13, V_(H) CDR 2.13, V_(H) CDR 3.13,V_(L) CDR 1.13, V_(L) CDR 2.13, and V_(L) CDR 3.13.
 14. The monoclonalantibody of claim 9, which is humanized.
 15. The monoclonal antibody ofclaim 14 which comprises V_(H) and V_(L) chains having sequencesselected from one of the following groups of V_(H) and V_(L) sequences:(i) hV_(H) 8a (SEQ ID NO:75) and hV_(L) 8a (SEQ ID NO:76); (ii) hV_(H)8b (SEQ ID NO:77) and hV_(L) 8b (SEQ ID NO:78); (iii) hV_(H) 8c (SEQ IDNO:79) and hV_(L) 8c (SEQ ID NO:76); (iv) hV_(H) 13a (SEQ ID NO:80) andhV_(L) 13a (SEQ ID NO:81); and (v) hV_(H) 13b (SEQ ID NO:82) and hV_(L)13b (SEQ ID NO:83).
 16. The monoclonal antibody of claim 1 which has anaffinity for hPG in the range of about 1 pM to about 7 nM.
 17. Themonoclonal antibody of claim 1 that binds to an epitope comprising anamino acid sequence corresponding to a sequence selected from the groupconsisting of SEQ ID NOs:28, 29, 30, 31, and
 32. 18. The monoclonalantibody of claim 1 that binds to an epitope comprising an amino acidsequence corresponding to a sequence selected from the group consistingof SEQ ID NOs:33, 34, 35, and
 36. 19. The monoclonal antibody of claim 1which competes for binding hPG with a reference antibody selected fromthe group consisting of: anti-hPG MAb1, anti-hPG MAb2, anti-hPG MAb3,anti-hPG MAb4, anti-hPG MAb15, anti-hPG MAb16, anti-hPG MAb17, anti-hPGMAb18, anti-hPG MAb19, and anti-hPG MAb20.
 20. The monoclonal antibodyof claim 1 which competes for binding hPG with a reference antibodyselected from the group consisting of: anti-hPG MAb5, anti-hPG MAb6,anti-hPG MAb7, anti-hPG MAb8, anti-hPG MAb9, anti-hPG MAb10, anti-hPGMAb11, anti-hPG MAb12, anti-hPG MAb13, anti-hPG MAb14, anti-hPG MAb21,anti-hPG MAb22, and anti-hPG MAb23.
 21. The monoclonal antibody of claim1 which neutralizes hPG activity in an in vitro assay carried out withLS174T cells.
 22. A composition comprising a monoclonal antibodyaccording to claim 1 and an excipient, carrier, and/or diluent.
 23. Thecomposition of claim 22 which is formulated for pharmaceutical use. 24.A polynucleotide encoding a variable light chain for an anti-hPGmonoclonal antibody according to claim
 1. 25. A polynucleotide encodinga variable heavy chain for an anti-hPG monoclonal antibody according toclaim
 1. 26. An expression vector comprising a polynucleotide encoding avariable light chain for an anti-hPG monoclonal antibody according toclaim
 1. 27. An expression vector comprising a polynucleotide encoding avariable heavy chain for an anti-hPG monoclonal antibody according toclaim
 1. 28. A host cell transformed with pairs of polynucleotidessuitable for expressing an anti-hPG monoclonal antibody according toclaim
 1. 29. A hybridoma capable of secreting an anti-hPG monoclonalantibody according to claim
 1. 30. The hybridoma of claim 29 selectedfrom the group consisting of: 43B9G11, WE5H2G7, 6B5B11C10, 20D2C3G2,1B4A11D11, 1B6A11F2, 1B11E4B11, 1C10D3B9, 1D8F5B3, 1E1C7B4, 2B4C8C8,2B11E6G4, 2C6C3C7, 2H9F4B7, 1E9A4A4, 1E9D9B6, 1C8D10F5, 1A7C3F11,1B3B4F11, 1C11F5E8, 1F11F5E10, 1F11F5G9, and 1A11F2C9.
 31. The hybridomaof claim 29, which is selected from the group consisting of: 43B9G11,WE5H2G7, 1B4A11D11, 1B6A11F2, 1B11E4B11, 1D8F5B3, 1E1C7B4, 2B4C8C8,2B11E6G4, 2H9F₄B7, 1E9A4A4, 1C8D10F5, 1A7C3F11, 1C11F5E8, 1F11F5E10,1F11F5G9, and 1A11F2C9.
 32. The hybridoma of claim 29, which is selectedfrom the group consisting of: 1B4A11D11, 1B6A11F2, 1B11E4B11, 2B4C8C8,2B11E6G4, and 1E9A4A4.
 33. A method of obtaining an anti-hPG monoclonalantibody comprising: (a) culturing a hybridoma of claim 21 undersuitable conditions; and (b) recovering the anti-hPG monoclonal antibodyfrom the culture medium or the hybridoma cells.
 34. A method of treatingcolorectal cancer comprising administering to a subject an amount of ahumanized neutralizing anti-hPG monoclonal antibody effective to treatthe colorectal cancer.
 35. The method of claim 34 in which theneutralizing anti-hPG monoclonal antibody competes for binding with ananti-hPG monoclonal antibody obtainable from a hybridoma selected fromthe group of consisting of: 43B9G11, WE5H2G7, 6B5B11C10, 20D2C3G2,1B4A11D11, 1B6A11F2, 1B11E4B11, 1C10D3B9, 1D8F5B3, 1E1C7B4, 2B4C8C8,2B11E6G4, 2C6C3C7, 2H9F4B7, 1E9A4A4, 1E9D9B6, 1C8D10F5, 1A7C3F11,1B3B4F11, 1C11F5E8, 1F11F5E10, 1F11F5G9, and 1A11F2C9.
 36. The method ofclaim 34 in which the neutralizing anti-hPG monoclonal antibody has CDRscorresponding in sequence to the CDRs of an anti-hPG monoclonal antibodyselected from the group consisting of: anti-hPG MAb1, anti-hPG MAb2,anti-hPG MAb3, anti-hPG MAb4, anti-hPG MAb5, anti-hPG MAb6, anti-hPGMAb7, anti-hPG MAb8, anti-hPG MAb9, anti-hPG MAb10, anti-hPG MAb11,anti-hPG MAb12, anti-hPG MAb13, anti-hPG MAb15, anti-hPG MAb16, anti-hPGMAb17, anti-hPG MAb18, anti-hPG MAb19, anti-hPG MAb20, anti-hPG MAb21,anti-hPG MAb22, or anti-hPG MAb23.
 37. A method of inhibitingproliferation of colorectal tumor cells, comprising exposing thecolorectal tumor cells to a neutralizing anti-hPG monoclonal antibody.38. The method of claim 37 in which the neutralizing anti-hPG monoclonalantibody competes for binding with an anti-hPG monoclonal antibodyobtainable from a hybridoma selected from the group of consisting of:43B9G11, WE5H2G7, 6B5B11C10, 20D2C3G2, 1B4A11D 11, 1B6A11F2, 1B11E4B11,1C10D3B9, 1D8F5B3, 1E1C7B4, 2B4C8C8, 2B11E6G4, 2C6C3C7, 1E9A4A4,1E9D9B6, 1C8D10F5, 1A7C3F11, 1B3B4F11, 1C11F5E8, 1F11F5E10, 1F11F5G9,and 1A11F2C9.
 39. The method of claim 37 in which the neutralizinganti-hPG monoclonal antibody has CDRs corresponding in sequence to theCDRs of an anti-hPG monoclonal antibody selected from the groupconsisting of anti-hPG MAb1, anti-hPG MAb2, anti-hPG MAb3, anti-hPGMAb4, anti-hPG MAb5, anti-hPG MAb6, anti-hPG MAb7, anti-hPG MAb8,anti-hPG MAb9, anti-hPG MAb10, anti-hPG MAb11, anti-hPG MAb12, anti-hPGMAb13, anti-hPG MAb15, anti-hPG MAb16, anti-hPG MAb17, anti-hPG MAb18,anti-hPG MAb19, anti-hPG MAb20, anti-hPG MAb21, anti-hPG MAb22, oranti-hPG MAb23.
 40. The method of claim 37 which is carried out invitro.
 41. The method of claim 37 which is carried out in vivo.
 42. Akit useful for detecting hPG, comprising an N-terminal anti-hPGmonoclonal antibody according to claim 2 and an antibody thatspecifically binds a C-terminal region of hPG.
 43. The kit of claim 42,wherein the antibody that specifically binds a C-terminal region of hPGis a polyclonal antibody.
 44. A kit useful for detecting hPG, comprisinga C-terminal anti-hPG monoclonal antibody according to claim 9 and anantibody that specifically binds an N-terminal region of hPG.
 45. Thekit of claim 44, wherein the antibody that specifically binds anN-terminal region of hPG is a polyclonal antibody.
 46. A method ofdiagnosing colorectal cancer in a subject, comprising quantifying theamount of progastrin in a serum sample from the subject using at leastone monoclonal antibody according to claim 1, wherein a quantity ofprogastrin in a range of about 20 to 400 pM is indicative of colorectalcancer.
 47. A method of selecting a patient suitable for anti-hPGtherapy, comprising quantifying the amount of progastrin in a serumsample from a patient with colorectal cancer using at least onemonoclonal antibody according to claim 1, wherein a quantity ofprogastrin in a range of about 20 to 400 pM is indicative of suitabilityfor anti-hPG therapy.
 48. A method of monitoring the effectiveness of acolorectal cancer treatment, comprising quantifying the amount ofprogastrin in a serum sample from a subject who is being treated forcolorectal cancer, as a function of time, using at least one monoclonalantibody according to claim 1, where a time-dependent decrease in thesubject's serum progastrin level indicates the treatment is effective.49. A method of inhibiting a progastrin-dependent signal transductioncascade in a cell, comprising exposing cells that secretes hPG to anamount of a neutralizing anti-hPG monoclonal antibody effective toinhibit progastrin-dependent signal transduction.